Compositions containing plant-produced glycopolypeptide multimers, multimeric proteins and method of their use

The present invention contemplates glycopolypeptide multimers having a polypeptide that contain an immunoglobulin amino acid residue sequence and an oligosaccharide that comprises a core pentasaccharide and N-acetylglucosamine-containing outer branches, such that the multimer is free from sialic acid. The production of passive immunity in an animal by administering a sialic acid free glycopolypeptide multimer is also contemplated. In addition, the invention describes a method for producing a glycopolypeptide multimer by introducing first and second mammalian genes encoding the constituent parts of the multimer into first and second respective members of a plant species, generating a progeny from the first and second plant species members, and isolating the glycopolypeptide multimer from the progeny plant.

TECHNICAL FIELD 
The present invention relates to expression of foreign multimeric proteins 
in plants as well as to transgenic plants that express such multimeric 
proteins. 
BACKGROUND 
It is known that polypeptides can be expressed in a wide variety of 
cellular hosts. A wide variety of structural genes have been isolated from 
mammals and viruses, joined to transcriptional and translational 
initiation and termination regulatory signals from a source other than the 
structural gene, and introduced into hosts into which these regulatory 
signals are functional. 
For economic reasons it would be desirable to utilize genetically 
engineered unicellular microorganisms to produce a wide variety of 
polypeptides. However, because of the inherent differences in the nature 
of unicellular organisms on one hand and mammalian cells on the other, the 
folding and processing of polypeptides in unicellular microorganisms 
appears to be quite different from the folding and processing that is 
effected in mammalian cells. As a result, mammalian polypeptides derived 
from unicellular microorganisms are not always properly folded or 
processed to provide the desired degree of biological or physiological 
activity in the obtained polypeptide. 
To that end attempts have been made, with varying degrees of success, to 
express mammalian polypeptides in plants. 
The first transgenic plants expressing foreign genes were tobacco plants 
produced by the use of Agrobacterium tumefaciens vectors as described by 
Horsch et al., Science, 223:496 (1984) and De Block et al., EMBO J., 
31:681 (1984). The transgenic plants produced in these studies were 
resistant to an antibiotic by virtue of the introduced transgene. These 
first transgenic plants were produced by introducing the foreign genes 
into plant protoplasts, i.e., plant cells that have had their cell wall 
removed either by mechanical action or enzymatic digestion. Subsequent 
transformation methods based on regenerable explants such as leaves, stems 
and roots have allowed the transformation of several dicotyledonous plant 
species. A variety of free DNA delivery methods, including microinjection, 
electroporation and particle gun technologies have allowed the 
transformation of monocotyledonous plants, such as corn, and rice. 
Expression of a multimeric protein, i.e., a protein constituted by two 
different polypeptide chains in significant yields has not been achieved. 
The factors controlling the assembly of such multimeric proteins are not 
well characterized except that it is known that the individual polypeptide 
chains must be present in sufficient quantity and in the same subcellular 
compartment. 
The expression of a multimeric protein in plant cells requires that the 
genes coding for the polypeptide chains be present in the same plant cell. 
The probability of actually introducing both genes into the same cell is 
extremely remote. Even if such a single cell cotransformant is produced, 
the plant must then be regenerated from this single-cell cotransformant. 
Multimeric proteins containing carbohydrate residues, glycopolypeptide 
multimers, are found in both plants and animals. Both plant and animal 
glycopolypeptide multimers have a common pentasaccharide core, 
Man.alpha.1-3(Man.alpha.1-6)Man.beta.1-4GLcNAc.beta.1-4-GLcNAc-, that is 
directly linked to an asparagine reside present in the polypeptide. See, 
Kornfeld and Kornfeld, Ann. Rev. Biochem., 54:631 (1985). 
Plant and animal glycopolypeptide multimers also have outer branches of 
oligosaccharides directly linked to the common pentasaccharide core. 
Animal and plant glycopolypeptide outer branches have N-acetylglucosamine 
in them, while the outer branches present in yeast only contain mannose. 
Plant and animal glycopolypeptide multimers contain different terminal 
carbohydrates that are directly linked to the outer branches of the 
oligosaccharides present. Animal glycopolypeptide multimers including 
mammalian glycopolypeptide multimers have sialic acid present as a 
terminal carbohydrate residue, while plant glycopolypeptide multimers do 
not. See Sturm et al., J. Biol. Chem., 262:13392 (1987). 
Secretory IgA is a glycopolypeptide multimer made up of two IgA molecules 
joining chain (J chain) and secretory component. IgA is the major class of 
antibody found in secretion, such as milk, saliva, tears, respiratory 
secretions and intestinal secretions. 
Secretory IgA is resistant to denaturation caused by harsh environments. 
This denaturation resistance requires that the complex secretory IgA 
molecule containing IgA molecules, J chain and secretory component be 
accurately and efficiently assembled. 
It has now been found that multimeric proteins can be expressed in 
relatively high yields in transgenic plants generated in a particular 
manner. In addition, the accurate and efficient expression of 
glycopolypeptide multimers free from sialic acid in transgenic plants has 
been discovered. 
It has also been discovered that passive immunity can be produced in an 
animal by providing sialic acid-containing soluble immunoglobulin 
containing a variety of different antibodies having varying antigen 
specificities to that animal. See, Tacket et al., New England J. Med., 
318:1240 (1988) and Eibl et al., New England J. Med., 319:1-7 (1988). To 
date there is no report of producing passive immunity in an animal by 
administering an encapsulated glycopolypeptide multimer capable of binding 
a preselected pathogen. 
A method of producing passive immunity by administering an encapsulated, 
glycopolypeptide multimer that is free from sialic acid and capable of 
binding a preselected pathogen has been discovered. 
SUMMARY OF THE INVENTION 
The production of biologically or physiologically active multimeric 
proteins such as abzymes, immunoglobulins, enzymes, and the like, in 
relatively high yields is achieved in a transgenic, sexually reproducible 
plant constituted by plant cells that each contain integrated within the 
nuclear genome plural mammalian genes coding for autogenously linking 
polypeptides as well as the autogenously linking polypeptides themselves. 
These polypeptides are present in the plant cells as a biologically active 
polypeptide multimer such as a homomultimer or a heteromultimer. These 
transgenic plants are morphologically normal but for the presence of the 
mammalian genes in substantially all of their cells. The respective gene 
products can be present in substantially all or a portion of the plant 
cells, i.e., the products can be localized to a cell type, tissue or 
organ. 
The foregoing transgenic plants are produced by introducing into the 
nuclear genome of a first member of the plant species a first mammalian 
gene that codes for an autogenously linkable monomeric polypeptide which 
is a constituent part of the multimeric protein to produce a viable first 
transformant. Similarly, another mammalian gene, coding for another 
autogenously linkable monomeric polypeptide which also is a constituent 
part of the multimeric protein is introduced into the nuclear genome of a 
second member of the same plant species to produce a viable second 
transformant. The so-obtained first and second transformants are then 
sexually crossed and cultivated to generate a progeny population from 
which transgenic plant species that produce the multimeric protein are 
isolated. 
Transgenic plants embodying the present invention are useful not only to 
produce economically, and in relatively high yields, the desired 
multimeric protein but also as means for separating and/or concentrating a 
preselected ligand, such as a metal ion, from a fluid, i.e., gas or 
liquid. 
The transgenic plants produce a glycopolypeptide multimer containing a 
polypeptide having a glycolsylated core portion as well as 
N-acetylglucosamine containing outer ranches and an amino acid residue 
sequence of an immunoglobulin molecule, where the multimer is free from 
detectable sialic acid residues. 
Passive immunity against a preselected pathogen is achieved in an animal by 
administering to the animal an encapsulated, biologically active 
glycopolypeptide multimer capable of binding a pathogen antigen in an 
amount sufficient to establish within said animal a prophylactic 
concentration of the multimer. The glycopolypeptide multimer administered 
is free from detectable sialic acid residues and contains a polypeptide 
having a glycosylated core portion as well as N-acetylglycosamine 
containing outer branches and an amino acid residue sequence of an 
immunoglobulin molecule. 
The present invention also contemplates biologically active compositions 
comprising a glycopolypeptide multimer containing a polypeptide having a 
glycosylated core portion as well as a N-acetylglucosamine containing 
outer branches and an amino acid residue sequence of an immunoglobulin 
molecule, where the multimer is free from detectable sialic acid residues 
and is encapsulated in a protective coating such as a plant cell.

DETAILED DESCRIPTION OF THE INVENTION 
A. Definitions 
Dicotyledon (dicot): A flowering plant whose embryos have two seed halves 
or cotyledons. Examples of dicots are: tobacco; tomato; the legumes 
including alfalfa; oaks; maples; roses; mints; squashes; daisies; walnuts; 
cacti; violets; and buttercups. 
Monocotyledon (monocot): A flowering plant whose embryos have one cotyledon 
or seed leaf. Examples of monocots are: lilies; grasses; corn; grains, 
including oats, wheat and barley; orchids; irises; onions and palms. 
Lower plant: Any non-flowering plant including ferns, gymnosperms, 
conifers, horsetails, club mosses, liver warts, hornworts, mosses, red 
algaes, brown algaes, gametophytes, sporophytes of pteridophytes, and 
green algaes. 
Eukaryotic hybrid vector: A DNA by means of which a DNA coding for a 
polypeptide (insert) can be introduced into a eukaryotic cell. 
Extrachromosomal ribosomal DNA (rDNA): A DNA found in unicellular 
eukaryotes outside the chromosomes, carrying one or more genes coding for 
ribosomal RNA and replicating autonomously (independent of the replication 
of the chromosomes). 
Palindromic DNA: A DNA sequence with one or more centers of symmetry. 
DNA: Deoxyribonucleic acid. 
T-DNA: A segment of transferred DNA. 
rDNA: Extrachromosomal Ribosomal DNA. 
RNA: Ribonucleic acid. 
rRNA: Ribosomal RNA. 
Ti-plasmid: Tumor-inducing plasmid. 
Ti-DNA: A segment of DNA from Ti-plasmid. 
Insert: A DNA sequence foreign to the host, consisting of a structural gene 
and optionally additional DNA sequences. 
Structural gene: A gene coding for a polypeptide and being equipped with a 
suitable promoter, termination sequence and optionally other regulatory 
DNA sequences, and having a correct reading frame. 
Signal Sequence: A DNA sequence coding for an amino acid sequence attached 
to the polypeptide which binds the polypeptide to the endoplasmic 
reticulum and is essential for protein secretion. 
(Selective) Genetic marker: A DNA sequence coding for a phenotypical trait 
by means of which transformed cells can be selected from untransformed 
cells. 
Promoter: A recognition site on a DNA sequence or group of DNA sequences 
that provide an expression control element for a gene and to which RNA 
polymerase specifically binds and initiates RNA synthesis (transcription) 
of that gene. 
Inducible promoter: A promoter where the rate of RNA polymerase binding and 
initiation is modulated by external stimuli. Such stimuli include light, 
heat, anaerobic stress, alteration in nutrient conditions, presence or 
absence of a metabolite, presence of a ligand, microbial attack, wounding 
and the like. 
Viral promoter: A promoter with a DNA sequence substantially similar to the 
promoter found at the 5' end of a viral gene. A typical viral promoter is 
found at the 5' end of the gene coding for the p2I protein of MMTV 
described by Huang et al., Cell, 27:245 (1981). (All references cited in 
this application are incorporated by reference.) 
Synthetic promoter: A promoter that was chemically synthesized rather than 
biologically derived. Usually synthetic promoters incorporate sequence 
changes that optimize the efficiency of RNA polymerase initiation. 
Constitutive promoter: A promoter where the rate of RNA polymerase binding 
and initiation is approximately constant and relatively independent of 
external stimuli. Examples of constitutive promoters include the 
cauliflower mosaic virus 35S and 19S promoters described by Poszkowski et 
al., EMBO J., 3:2719 (1989) and Odell et al., Nature, 313:810 (1985). 
Temporally regulated promoter: A promoter where the rate of RNA polymerase 
binding and initiation is modulated at a specific time during development. 
Examples of temporally regulated promoters are given in Chua et al., 
Science, 244:174-181 (1989). 
Spatially regulated promoter: A promoter where the rate of RNA polymerase 
binding and initiation is modulated in a specific structure of the 
organism such as the leaf, stem or root. Examples of spatially regulated 
promoters are given in Chua et al., Science, 244:174-181 (1989). 
Spatiotemporally regulated promoter: A promoter where the rate of RNA 
polymerase binding and initiation is modulated in a specific structure of 
the organism at a specific time during development. A typical 
spatiotemporally regulated promoter is the EPSP synthase-35S promoter 
described by Chua et al., Science, 244:174-181 (1989). 
Single-chain antigen-binding protein: A polypeptide composed of an 
immunoglobulin light-chain variable region amino acid sequence (V.sub.L) 
tethered to an immunoglobulin heavy-chain variable region amino acid 
sequence (V.sub.H) by a peptide that links the carboxyl terminus of the 
V.sub.L sequence to the amino terminus of the V.sub.H sequence. 
Single-chain antigen-binding protein-coding gene: A recombinant gene coding 
for a single-chain antigen-binding protein. 
Multimeric protein: A globular protein containing more than one separate 
polypeptide or protein chain associated with each other to form a single 
globular protein. Both heterodimeric and homodimeric proteins are 
multimeric proteins. 
Polypeptide and peptide: A linear series of amino acid residues connected 
one to the other by peptide bonds between the alpha-amino and carboxy 
groups of adjacent residues. 
Protein: A linear series of greater than about 50 amino acid residues 
connected one to the other as in a polypeptide. 
Chelating agent: A chemical compound, peptide or protein capable of binding 
a metal. Examples of chelating agents include ethylene diamine tetra 
acetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether) N, N, N', 
N'-tetraacetic acid (EGTA), 2,3-dimercaptopropanol-1-sulfonic acid (DMPS), 
and 2,3-dimercaptosuccinic acid (DMSA), and the like. 
Metal chelation complex: A complex containing a metal bound to a chelating 
agent. 
Immunoglobulin product: A polypeptide, protein or multimeric protein 
containing at least the immunologically active portion of an 
immunoglobulin heavy chain and is thus capable of specifically combining 
with an antigen. Exemplary immunoglobulin products are an immunoglobulin 
heavy chain, immunoglobulin molecules, substantially intact immunoglobulin 
molecules, any portion of an immunoglobulin that contains the paratope, 
including those portions known in the art as Fab fragments, Fab' fragment, 
F(ab').sub.2 fragment and Fv fragment. 
Immunoglobulin molecule: A multimeric protein containing the 
immunologically active portions of an immunoglobulin heavy chain and 
immunoglobulin light chain covalently coupled together and capable of 
specifically combining with antigen. 
Fab fragment: A multimeric protein consisting of the portion of an 
immunoglobulin molecule containing the immunologically active portions of 
an immunoglobulin heavy chain and an immunoglobulin light chain covalently 
coupled together and capable of specifically combining with antigen. Fab 
fragments are typically prepared by proteolytic digestion of substantially 
intact immunoglobulin molecules with papain using methods that are well 
known in the art. However, a Fab fragment may also be prepared by 
expressing in a suitable host cell the desired portions of immunoglobulin 
heavy chain and immunoglobulin light chain using methods well known in the 
art. 
F.sub.v fragment: A multimeric protein consisting of the immunologically 
active portions of an immunoglobulin heavy chain variable region and an 
immunoglobulin light chain variable region covalently coupled together and 
capable of specifically combining with antigen. F.sub.v fragments are 
typically prepared by expressing in suitable host cell the desired 
portions of immunoglobulin heavy chain variable region and immunoglobulin 
light chain variable region using methods well known in the art. 
Asexual propagation: Producing progeny by regenerating an entire plant from 
leaf cuttings, stem cuttings, root cuttings, single plant cells 
(protoplasts) and callus. 
Glycosylated core portion: The pentasaccharide core common to all 
asparagine-linked oligosaccharides. The pentasaccharide care has the 
structure Man.alpha.1-3(man.alpha.1-6) Man.beta.1-46LcNAc.beta.1-4 
6LcNac-(ASN amino acid). The pentasaccharide core typically has 2 outer 
branches linked to the pentasaccharide core. 
N-acetylglucosamine containing outer branches: The additional 
oligosaccharides that are linked to the pentasaccharide core (glycosylated 
core portion) of asparagine-linked oligosaccharides. The outer branches 
found on both mammalian and plant glycopolypeptides contain 
N-acetylglucosamine in direct contrast with yeast outer branches that only 
contain mannose. Mammalian outer branches have sialic acid residues linked 
directly to the terminal portion of the outer branch. 
Glycopolypeptide multimer: A globular protein containing a glycosylated 
polypeptide or protein chain and at least one other polypeptide or protein 
chain bonded to each other to form a single globular protein. Both 
heterodimeric and homodimeric glycoproteins are multimeric proteins. 
Glycosylated polypeptides and proteins are n-glycans in which the C(I) of 
N-acetylglucosamine is linked to the amide group of asparagine. 
Immunoglobulin superfamily molecule: A molecule that has a domain size and 
amino acid residue sequence that is significantly similar to 
immunoglobulin or immunoglobulin related domains. The significance of 
similarity is determined statistically using a computer program such as 
the Align program described by Dayhoff et al., Meth Enzymol., 91:524-545 
(1983). A typical Align score of less than 3 indicates that the molecule 
being tested is a member of the immunoglobulin gene superfamily. 
The immunoglobulin gene superfamily contains several major classes of 
molecules including those shown in Table A and described by Williams and 
Barclay, in Immunoglobulin Genes, p361, Academic Press, New York, N.Y. 
(1989). 
TABLE A 
______________________________________ 
The Known Members of The Immunoglobulin Gene Superfamily* 
______________________________________ 
Immunoglobulin 
Heavy chains (IgM) 
Light chain kappa 
Light chain lambda 
T cell receptor (Tcr) complex 
Tcr .alpha.-chain 
Tcr .beta. chain 
Tcr gamma chain 
Tcr X-chain 
CD3 gamma chain 
CD3 .delta.-chain 
CD3 .epsilon.-chain 
Major histocompatibility complex (MHC) antigens 
Class I H-chain 
.beta..sub.2 -microglobulin 
Class II .alpha. 
Class II .beta. 
.beta..sub.2 -m associated antigens 
TL H chain 
Qa-2 H chain 
CD1a H chain 
T lymphocyte antigens 
CD2 
CD4 
CD7 
CD8 chain I 
CD8 Chain IId 
CD28 
CTLA4 
Haemopoietic/endothelium antigens 
LFA-3 
MRC OX-45 
Brain/lymphoid antigens 
Thy-1 
MRC OX-2 
Immunoglobulin receptors 
Poly Ig R 
Fc gamma 2b/gamma 1R 
Fc.epsilon.RI(.alpha.) 
Neural molecules 
Neural adhesion molecule (MCAM) 
Myelin associated gp (MAG) 
P.sub.0 myelin protein 
Tumor antigen 
Carcinoembryonic antigen (CEA) 
Growth factor receptors 
Platelet-derived growth factor (PDGF) receptor 
Colony stimulating factor-1 (CSF1) receptor 
Non-cell surface molecules 
.alpha..sub.1 B-glycoprotein 
Basement membrane link protein 
______________________________________ 
*See Williams and Barclay, in Immunglobulin Genes, p 361, Academic Press, 
NY (1989); and Sequences of Proteins of Immunological Interest, 4th ed., 
U.S. Dept. of Health and Human Serving (1987). 
Catalytic site: The portion of a molecule that is capable of binding a 
reactant and improving the rate of a reaction. Catalytic sites may be 
present on polypeptides or proteins, enzymes, organics, organo-metal 
compounds, metals and the like. A catalytic site may be made up of 
separate portions present on one or more polypeptide chains or compounds. 
These separate catalytic portions associate together to form a larger 
portion of a catalytic site. A catalytic site may be formed by a 
polypeptide or protein that is bonded to a metal. 
Enzymatic site: The portion of a protein molecule that contains a catalytic 
site. Most enzymatic sites exhibit a very high selective substrate 
specificity. An enzymatic site may be comprised of two or more enzymatic 
site portions present on different segments of the same polypeptide chain. 
These enzymatic site portions are associated together to form a greater 
portion of an enzymatic site. A portion of an enzymatic site may also be a 
metal. 
Self-pollination: The transfer of pollen from male flower parts to female 
flower parts on the same plant. This process typically produces seed. 
Cross-pollination: The transfer of pollen from the male flower parts of one 
plant to the female flower parts of another plant. This process typically 
produces seed from which viable progeny can be grown. 
Epitope: A portion of a molecule that is specifically recognized by an 
immunoglobulin product. It is also referred to as the determinant or 
antigenic determinant. 
Abzyme: An immunoglobulin molecule capable of acting as an enzyme or a 
catalyst. 
Enzyme: A protein, polypeptide, peptide RNA molecule, or multimeric protein 
capable of accelerating or producing by catalytic action some change in a 
substrate for which it is often specific. 
B. Methods of Producing Transgenic Plants Containing A Multimeric Protein 
The present invention provides a novel method for producing a plant 
containing a multimeric protein comprised of first and second 
polypeptides. Generally, the method combines the following elements: 
1. Inserting into the genome of a first member of a plant species a gene 
coding for a first polypeptide to produce a first transformant. 
2. Inserting into the genome of a second member of a plant species a gene 
coding for a second polypeptide to produce a second transformant. 
3. Producing a population of progeny from the first and second 
transformants. 
4. Isolating from the population, a progeny having the multimeric protein. 
A plant produced by the present invention contains a multimeric protein 
comprised of a first and second polypeptide associated together in such a 
way as to assume a biologically functional conformation. In one embodiment 
of this invention, the multimeric protein is a ligand binding polypeptide 
(receptor) that forms a ligand binding site which specifically binds to a 
preselected ligand to form a complex having a sufficiently strong binding 
between the ligand and the ligand binding site for the complex to be 
isolated. In another embodiment, the multimeric protein is an 
immunoglobulin molecule comprised of an immunoglobulin heavy chain and an 
immunoglobulin light chain. The immunoglobulin heavy and light chains are 
associated with each other and assume a conformation having an antigen 
binding site specific for, as evidenced by its ability to be competitively 
inhibited, a preselected or predetermined antigen. When the multimeric 
protein is an antigen binding protein its affinity or avidity is generally 
greater than 10.sup.5 M.sup.-1 or usually greater than 10.sup.6 M.sup.-1, 
and preferably greater than 10.sup.8 M.sup.-1. 
In a further embodiment, the multimeric protein is a Fab fragment 
consisting of a portion of an immunoglobulin heavy chain and a portion of 
an immunoglobulin light chain. The immunoglobulin heavy and light chains 
are associated with each other and assume a conformation having an antigen 
binding site specific for a preselected or predetermined antigen. The 
antigen binding site on a Fab fragment has a binding affinity or avidity 
similar to the antigen binding site on an immunoglobulin molecule. 
In yet another embodiment, the present transgenic plant contains a 
multimeric protein that is a F.sub.v fragment comprised of at least a 
portion of an immunoglobulin heavy chain variable region and at least a 
portion of an immunoglobulin light chain variable region. The 
immunoglobulin heavy and light chain variable regions autogenously 
associate with each other within the plant cell to assume a biologically 
active conformation having a binding site specific for a preselected or 
predetermined antigen. The antigen binding site on the Fv fragment has an 
affinity or avidity for its antigen similar to the affinity displayed by 
the antigen binding site present on an immunoglobulin molecule. 
In still another embodiment, the multimeric protein is an enzyme that binds 
a substrate and catalyzes the formation of a product from the substrate. 
While the topology of the substrate binding site (ligand binding site) of 
the catalytic multimeric protein is probably more important for its 
activity than affinity (association constant or pKa) for the substrate, 
the subject multimeric protein has an association constant for its 
preselected substrate greater than 10.sup.3 M.sup.-1, more usually greater 
than 10.sup.5 M.sup.-1 or 10.sup.6 M.sup.-1 and preferably greater than 
10.sup.7 M.sup.-1. 
When the multimeric protein produced in accordance with the present 
invention is an abzyme comprised of at least a portion of the 
immunoglobulin heavy chain variable region in association with another 
polypeptide chain, this other polypeptide chain includes at least the 
biologically active portion of an immunoglobulin light chain variable 
region. Together, these two polypeptides assume a conformation having a 
binding affinity or association constant for a preselected ligand that is 
different, preferably higher, than the affinity or association constant of 
either of the polypeptides alone, i.e., as monomers. Useful multimeric 
proteins contain one or both polypeptide chains derived from the variable 
region of the light and heavy chains of an immunoglobulin. Typically, 
polypeptides comprising the light (V.sub.L) and heavy (V.sub.H) chain 
variable regions are employed together for binding the preselected 
antigen. 
1. Inserting Genes Coding For A First Polypeptide Into A First Member Of A 
Plant Species 
Methods for isolating a gene coding for a desired first polypeptide are 
well known in the art. See, for example, Guide To Molecular Cloning 
Techniques in Methods In Enzymology, Volume 152, Berger and Kimmel, eds. 
(1987); and Current Protocols in Molecular Biology, Ausubel et al., eds., 
John Wiley and Sons, New York (1987) whose disclosures are herein 
incorporated by reference. 
Genes useful in practicing this invention include gene coding for a 
polypeptide contained in immunoglobulin products, immunoglobulin 
molecules, Fab fragments, F.sub.v fragments, enzymes, receptors and 
abzymes. Particularly preferred are genes coding for immunoglobulin heavy 
and light chain variable regions. Typically, the genes coding for the 
immunoglobulin heavy chain variable region and immunoglobulin light chain 
variable region of an immunoglobulin capable of binding a preselected 
antigen are used. These genes are isolated from cells obtained from a 
vertebrate, preferably a mammal, which has been immunized with an 
antigenic ligand (antigen) against which activity is sought, i.e., a 
preselected antigen. The immunization can be carried out conventionally 
and antibody titer in the animal can be monitored to determine the stage 
of immunization desired, which corresponds to the affinity or avidity 
desired. Partially immunized animals typically receive only one 
immunization and cells are collected therefrom shortly after a response is 
detected. Fully immunized animals display a peak titer which is achieved 
with one or more repeated injections of the antigen into the host mammal, 
normally at two to three week intervals. 
Usually three to five days after the last challenge, the spleen is removed 
and the genes coding for immunoglobulin heavy and immunoglobulin light 
chain are isolated from the rearranged B cells present in the spleen using 
standard procedures. See Current Protocols in Molecular Biology, Ausubel 
et al., eds., John Wiley and Sons, New York (1987) and Antibodies: A 
Laboratory Manual, Harlowe and Lane, eds., Cold Spring Harbor, New York 
(1988). 
Genes coding for V.sub.H and V.sub.L polypeptides can be derived from cells 
producing IgA, IgD, IgE, IgG or IgM, most preferably from IgM and IgG, 
producing cells. Methods for preparing fragments of genomic DNA from which 
immunoglobulin variable region genes can be cloned are well known in the 
art. See for example, Herrmann et al., Methods in Enzymol., 152:180-183 
(1987); Frischauf, Methods in Enzymol., 152:183-190 (1987); Frischauf, 
Methods in Enzymol., 152:199-212 (1987). (The teachings of the references 
cited herein are hereby incorporated by reference). 
Probes useful for isolating the genes coding for immunoglobulin products 
include the sequences coding for the constant portion of the V.sub.H and 
V.sub.L sequences coding for the framework regions of V.sub.H and V.sub.L 
and probes for the constant region of the entire rearranged immunoglobulin 
gene, these sequences being obtainable from available sources. See for 
example, Early and Hood, Genetic Engineering, Setlow and Hollaender eds., 
Vol. 3:157-188, Plenum Publishing Corporation, New York (1981); and Kabat 
et al., Sequences of Immunological Interests, National Institutes of 
Health, Bethesda, Md. (1987). 
Genes coding for a polypeptide subunit of a multimeric protein can be 
isolated from either the genomic DNA containing the gene expressing the 
polypeptide or the messenger RNA (mRNA) which codes for the polypeptide. 
The difficulty in using genomic DNA is in juxtaposing the sequences coding 
for the polypeptide where the sequences are separated by introns. The DNA 
fragment(s) containing the proper exons must be isolated, the introns 
excised, and the exons spliced together in the proper order and 
orientation. For the most part, this will be difficult so the alternative 
technique employing mRNA will be the method of choice because the sequence 
is contiguous (free of introns) for the entire polypeptide. Methods for 
isolating mRNA coding for peptides or proteins are well known in the art. 
See, for example, Current Protocols in Molecular Biology, Ausubel et al., 
John Wiley and Sons, New York (1987), Guide to Molecular Cloning 
Techniques, in Methods In Enzymology, Volume 152, Berger and Kimmel, eds. 
(1987), and Molecular Cloning: A Laboratory Manual, Maniatis et al., eds., 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982). 
The polypeptide coding genes isolated above are typically operatively 
linked to an expression vector. Expression vectors compatible with the 
host cells, preferably those compatible with plant cells are used to 
express the genes of the present invention. Typical expression vectors 
useful for expression of genes in plants are well known in the art and 
include vectors derived from the tumorinducing (Ti) plasmid of 
Agrobacterium tumefaciens described by Rogers et al., Meth. in Enzymol., 
153:253-277 (1987). However, several other expression vector systems are 
known to function in plants. See for example, Verma et al., PCT 
Publication No. WO87/00551; and Cocking and Davey, Science, 236:1259-1262 
(1987). 
The expression vectors described above contain expression control elements 
including the promoter. The polypeptide coding genes are operatively 
linked to the expression vector to allow the promoter sequence to direct 
RNA polymerase binding and synthesis of the desired polypeptide coding 
gene. Useful in expressing the polypeptide coding gene are promoters which 
are inducible, viral, synthetic, constitutive, temporally regulated, 
spatially regulated, and spatiotemporally regulated. The choice of which 
expression vector and ultimately to which promoter a polypeptide coding 
gene is operatively linked depends directly, as is well known in the art, 
on the functional properties desired, e.g. the location and timing of 
protein expression, and the host cell to be transformed, these being 
limitations inherent in the art of constructing recombinant DNA molecules. 
However, an expression vector useful in practicing the present invention 
is at least capable of directing the replication, and preferably also the 
expression of the polypeptide coding gene included in the DNA segment to 
which it is operatively linked. 
In preferred embodiments, the expression vector used to express the 
polypeptide coding gene includes a selection marker that is effective in a 
plant cell, preferably a drug resistance selection marker. A preferred 
drug resistance marker is the gene whose expression results in kanamycin 
resistance, i.e., the chimeric gene containing the nopaline synthase 
promoter, Tn5 neomycin phosphotransferase II and nopaline synthase 3' 
nontranslated region described by Rogers et al., in Methods For Plant 
Molecular Biology, a Weissbach and H. Weissbach, eds., Academic Press 
Inc., San Diego, Calif. (1988 . A useful plant expression vector is 
commercially available from Pharmacia, Piscataway, N.J. 
A variety of methods have been developed to operatively link DNA to vectors 
via complementary cohesive termini. For instance, complementary 
homopolymer tracks can be added to the DNA segment to be inserted and to 
the vector DNA. The vector and DNA segment are then joined by hydrogen 
bonding between the complementary homopolymeric tails to form recombinant 
DNA molecules. 
Alternatively, synthetic linkers containing one or more restriction 
endonuclease sites can be used to join the DNA segment to the expression 
vector. The synthetic linkers are attached to blunt-ended DNA segments by 
incubating the blunt-ended DNA segments with a large excess of synthetic 
linker molecules in the presence of an enzyme that is able to catalyze the 
ligation of blunt-ended DNA molecules, such as bacteria phage T4 DNA 
ligase. Thus, the products of the reaction are DNA segments carrying 
synthetic linker sequences at their ends. These DNA segments are then 
cleaved with the appropriate restriction endonuclease and ligated into an 
expression vector that has been cleaved with an enzyme that produces 
termini compatible with those of the synthetic linker. Synthetic linkers 
containing a variety of restriction endonuclease sites are commercially 
available from a number of sources including New England BioLabs, Beverly, 
Mass. 
Methods for introducing polypeptide coding genes into plants include 
Agrobacterium-mediated plant transformation, protoplast transformation, 
gene transfer into pollen, injection into reproductive organs and 
injection into immature embryos. Each of these methods has distinct 
advantages and disadvantages. Thus, one particular method of introducing 
genes into a particular plant species may not necessarily be the most 
effective for another plant species. 
Agrobacterium tumefaciens-mediated transfer is a widely applicable system 
for introducing genes into plant cells because the DNA can be introduced 
into whole plant tissues, bypassing the need for regeneration of an intact 
plant from a protoplast. The use of Agrobacterium-mediated expression 
vectors to introduce DNA into plant cells is well known in the art. See, 
for example, the methods described by Fraley et al., Biotechnology, 3:629 
(1985) and Rogers et al., Methods in Enzymology, 153:253-277 (1987). 
Further, the integration of the Ti-DNA is a relatively precise process 
resulting in few rearrangements. The region of DNA to be transferred is 
defined by the border sequences and intervening DNA is usually inserted 
into the plant genome as described by Spielmann et al., Mol. Gen. Genet., 
205:34 (1986) and Jorgensen et al., Mol. Gen. Genet., 207:471 (1987). 
Modern Agrobacterium transformation vectors are capable of replication in 
Escherichia coli as well as Aorobacterium, allowing for convenient 
manipulations as described by Klee et al., in Plant DNA Infectious Agents, 
T. Hohn and J. Schell, eds., Springer-Verlag, New York (1985) pp. 179-203. 
Further recent technological advances in vectors for 
Agrobacterium-mediated gene transfer have improved the arrangement of 
genes and restriction sites in the vectors to facilitate construction of 
vectors capable of expressing various polypeptide coding genes. The 
vectors described by Rogers et al., Methods in Enzymology, 153:253 (1987), 
have convenient multilinker regions flanked by a promoter and a 
polyadenylation site for direct expression of inserted polypeptide coding 
genes and are suitable for present purposes. 
In those plant species where Agrobacterium-mediated transformation is 
efficient, it is the method of choice because of the facile and defined 
nature of the gene transfer. However, few monocots appear to be natural 
hosts for Aorobacterium. Although transgenic plants have been produced in 
asparagus using Agrobacterium vectors as described by Bytebier et al., 
Proc. Natl. Acad. Sci. U.S.A., 84:5345 (1987). Therefore, commercially 
important cereal grains such as rice, corn, and wheat must be transformed 
using alternative methods. Transformation of plant protoplasts can be 
achieved using methods based on calcium phosphate precipitation, 
polyethylene glycol treatment, electroporation, and combinations of these 
treatments. See, for example, Potrykus et al., Mol. Gen. Genet., 199:183 
(1985); Lorz et al., Mol. Gen. Genet., 199:178 (1985); Fromm et al., 
Nature, 319:791 (1986); Uchimiya et al., Mol. Gen. Genet., 204:204 (1986); 
Callis et al., Genes and Development, 1:1183 (1987); and Marcotte et al., 
Nature, 335:454 (1988). 
Application of these systems to different plant species depends upon the 
ability to regenerate that particular plant species from protoplasts. 
Illustrative methods for the regeneration of cereals from protoplasts are 
described in Fujimura et al., Plant Tissue Culture Letters, 2:74 (1985); 
Toriyama et al., Theor Appl. Genet., 73:16 (1986); Yamada et al., Plant 
Cell Reo., 4:85 (1986); Abdullah et al., Biotechnology, 4:1087 (1986). 
Agrobacterium-mediated transformation of leaf disks and other tissues 
appears to be limited to plant species that Aorobacterium tumefaciens 
naturally infects. Thus, Aorobacterium-mediated transformation is most 
efficient in dicotyledonous plants. However, the transformation of 
Asparagus using Agrobacterium can also be achieved. See, for example, 
Bytebier, et al., Proc. Natl. Acad. Sci., 84:5345 (1987). 
To transform plant species that cannot be successfully regenerated from 
protoplast, other ways to introduce DNA into intact cells or tissues can 
be utilized. For example, regeneration of cereals from immature embryos or 
explants can be effected as described by Dasil, Biotechnoloqv, 6:397 
(1988). In addition, "particle gun" or high-velocity microprojectile 
technology can be utilized as well. Using such technology, DNA is carried 
through the cell wall and into the cytoplasm on the surface of small 
(0.525 um) metal particles that have been accelerated to speeds of one to 
several hundred meters per second as described in Klein et al., Nature, 
327:70 (1987); Klein et al., Proc. Natl. Acad. Sci. U.S.A., 85:8502 
(1988); and McCabe et al., Biotechnology, 6:923 (1988). The metal 
particles penetrate through several layers of cells and thus allow the 
transformation of cells within tissue explants. Metal particles have been 
used to successfully transform corn cells and to produce fertile, stably 
transformed tobacco and soybean plants. Transformation of tissue explants 
eliminates the need for passage through a protoplast stage and thus speeds 
the production of transgenic plants. 
DNA can be introduced into plants also by direct DNA transfer into pollen 
as described by Zhou et al., Methods in Enzymology, 101:433 (1983); D. 
Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., Plant Mol. Biol. 
Reporter, 6:165 (1988). Expression of polypeptide coding genes can be 
obtained by injection of the DNA into reproductive organs of a plant as 
described by Pena et al., Nature, 325:274 (1987). DNA can also be injected 
directly into the cells of immature embryos and the rehydration of 
desiccated embryos as described by Neuhaus et al., Theor. Apl. Genet., 
75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, 
Butterworth, Stoneham, MA, pp. 27-54 (1986). 
The regeneration of plants from either single plant protoplasts or various 
explants is well known in the art. See, for example, Methods for Plant 
Molecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press, 
Inc., San Diego, Calif. (1988). This regeneration and growth process 
includes the steps of selection of transformant cells and shoots, rooting 
the transformant shoots and growth of the plantlets in soil. 
The regeneration of plants containing the foreign gene introduced by 
Agrobacterium tumefaciens from leaf explants can be achieved as described 
by Horsch et al., Science. 227:1229-1231 (1985). In this procedure, 
transformants are grown in the presence of a selection agent and in a 
medium that induces the regeneration of shoots in the plant species being 
transformed as described by Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 
80:4803 (1983). This procedure typically produces shoots within two to 
four weeks and these transformant shoots are then transferred to an 
appropriate root-inducing medium containing the selective agent and an 
antibiotic to prevent bacterial growth. Transformant shoots that rooted in 
the presence of the selective agent to form plantlets are then 
transplanted to soil to allow the production of roots. These procedures 
will vary depending upon the particular plant species employed, such 
variations being well known in the art. 
2. Inserting A Gene Coding For A Second Polypeptide Into A Second Member Of 
A Plant Species 
Useful genes include those genes coding for a second polypeptide that can 
autogenously associate with the first polypeptide in such a way as to form 
a biologically functional multimeric protein. The methods used to 
introduce a gene coding for this second polypeptide into a second member 
of a plant species are the same as the methods used to introduce a gene 
into the first member of the same plant species and have been described 
above. 
3. Producing A Population of Progeny From The First And Second 
Transformants 
A population of progeny can be produced from the first and second 
transformants of a plant species by methods well known in the art 
including those methods known as cross fertilization described by Mendel 
in 1865 (an English translation of Mendel's original paper together with 
comments and a bibliography of Mendel by others can be found in 
Experiments In Plant Hybridization, Edinburgh, Scotland, Oliver Boyd, 
eds., 1965). 
4. Isolating Progeny Containing The Multimeric Protein 
Progeny containing the desired multimeric protein can be identified by 
assaying for the presence of the biologically active multimeric protein 
using assay methods well known in the art. Such methods include Western 
blotting, immunoassays, binding assays, and any assay designed to detect a 
biologically functional multimeric protein. See, for example, the assays 
described in Immunology: The Science of Self-Nonself Discrimination, 
Klein, John Wiley and Sons, New York, N.Y. (1982). 
Preferred screening assays are those where the biologically active site on 
the multimeric protein is detected in such a way as to produce a 
detectible signal. This signal may be produced directly or indirectly and 
such signals include, for example, the production of a complex, formation 
of a catalytic reaction product, the release or uptake of energy, and the 
like. For example, a progeny containing an antibody molecule produced by 
this method may be processed in such a way to allow that antibody to bind 
its antigen in a standard immunoassay such as an ELISA or a 
radio-immunoassay similar to the immunoassays described in Antibodies: A 
Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y. (1988). 
A further aspect of the present invention is a method of producing a 
multimeric protein comprised of a first and a second polypeptide. 
Generally, the method combines the elements of cultivating a plant of the 
present invention, and harvesting the plant that was cultivated to produce 
the desired multimeric protein. 
A plant of the present invention containing the desired multimeric protein 
comprised of a first polypeptide and a second polypeptide is cultivated 
using methods well known to one skilled in the art. Any of the transgenic 
plants of the present invention may be cultivated to isolate the desired 
multimeric protein they contain. 
After cultivation, the transgenic plant is harvested to recover the 
produced multimeric protein. This harvesting step may consist of 
harvesting the entire plant, or only the leaves, or roots of the plant. 
This step may either kill the plant or if only the portion of the 
transgenic plant is harvested may allow the remainder of the plant to 
continue to grow. 
In preferred embodiments this harvesting step further comprises the steps 
of: 
(i) homogenizing at least a portion of said transgenic plant to produce a 
plant pulp; 
(ii) extracting said multimeric protein from said plant pulp to produce a 
multimeric protein containing solution; and 
(iii) isolating said multimeric protein from said solution. 
At least a portion of the transgenic plant is homogenized to produce a 
plant pulp using methods well known to one skilled in the art. This 
homogenization may be done manually, by a machine, or by a chemical means 
as long as the transgenic plant portions are broken up into small pieces 
to produce a plant pulp. This plant pulp consists of a mixture of varying 
sizes of transgenic plant particles. The size of the plant particles and 
the amount of variation in size that can be tolerated will depend on the 
exact method used to extract the multimeric protein from the plant pulp 
and these parameters are well known to one skilled in the art. 
The multimeric protein is extracted from the plant pulp produced above to 
form a multimeric protein containing solution. Such extraction processes 
are common and well known to one skilled in this art. For example, the 
extracting step may consist of soaking or immersing the plant pulp in a 
suitable solvent. This suitable solvent is capable of dissolving the 
multimeric protein present in the plant pulp to produce a multimeric 
protein containing solution. Solvents useful for such an extraction 
process are well known to those skilled in the art and include aqueous 
solvents, organic solvents and combinations of both. 
The multimeric protein is isolated from the solution produced above using 
methods that are well known to those skilled in the art of protein 
isolation. These methods include, but are not limited to, immuno-affinity 
purification and purification procedures based on the specific size, 
electrophoretic mobility, biological activity, and/or net charge of the 
multimeric protein to be isolated. 
C. Utilization of the Transgenic Plant 
The present invention also provides a novel method for separating a 
preselected ligand from a fluid sample. The method combines the following 
elements: 
1. Commingling the fluid sample with plant cells from a transgenic plant 
from the present invention to form an admixture. 
2. Maintaining this admixture for a time period sufficient for the ligand 
to enter the plant cells and bind the multimeric protein to form a complex 
within the plant cells. 
3. Removing the complex-containing plant cells from the admixture and 
thereby separating the ligand from the fluid sample. 
The fluid sample is commingled with the plant cells from a transgenic plant 
of the present invention that contain a multimeric protein. This 
multimeric protein can be a receptor, an enzyme, an immunoglobulin 
product, an immunoglobulin molecule or fragment thereof, or an abzyme. One 
skilled in the art will understand that this multimeric protein must be 
capable of binding the preselected ligand. The fluid sample can be a 
liquid or a gas. In either case the commingling may consist of placing the 
plant cells in either the liquid or the gas. Alternatively, the plant 
cells may be thoroughly mixed with the fluid sample. This commingling must 
bring the fluid sample in intimate contact with the plant cells to form an 
admixture. 
This admixture is maintained for a time period sufficient to allow the 
ligand present in the fluid sample to enter the cells. This process may be 
a passive process as in diffusion or may occur through the application of 
energy to the system, such as applying high pressure to the fluid sample 
to force it into the plant cells. The amount of time required for the 
ligand to enter the plant cells is known to one skilled in the art and can 
be predetermined to optimize such time period. After entering the plant 
cells the ligand binds the multimeric protein to form a complex. When the 
multimeric protein is a receptor, the complex formed is a receptor-ligand 
complex. When the multimeric protein is an immunoglobulin, immunoglobulin 
molecule, a portion of an immunoglobulin molecule, a Fab fragment, or a Fv 
fragment the complex formed is an immuno-reaction complex. When the 
multimeric protein is an enzyme and the ligand is a substrate the complex 
formed is an enzyme-substrate complex. When the multimeric protein is an 
abzyme the complex formed is an immuno-reaction complex. 
After the complex is formed in the plant cells, the complex-containing 
plant cells are removed from the admixture thereby separating the ligand 
from the fluid sample. Methods for removing the plant cells from the 
admixtures are well known to those skilled in the art and include 
mechanical removal, filtration, sedimentation and other separation means. 
When the plant cells utilized for this method constitute a viable plant, 
this expedient concentrates the ligand within the plant. When the ligand 
is an important nutrient, this results in that plant concentrating that 
particular nutrient within its cells, thereby enhancing the nutritional 
value of the plant. When the ligand is an environmental pollutant, this 
pollutant is concentrated within the plant cells and thus is removed from 
the environment. Of course, for this method to be applicable, the ligand 
must be able to enter the plant cells. The ligands that can enter the 
plant cells are well known to those skilled in the art. 
The present invention also contemplates a method of separating a metal ion 
from a fluid sample containing the metal ion. This particular method 
includes the following steps: 
1. Admixing to the fluid sample a chelating agent to form a chelating 
admixture. 
2. Maintaining the chelating admixture for a time period sufficient for the 
metal ion to bind the chelating agent and form a metal ion chelation 
complex. 
3. Commingling the metal ion chelation complex with plant cells of the 
present invention to form a binding admixture. 
4. Maintaining the binding admixture for a time period sufficient for the 
metal ion chelation complex to enter the plant cells and bind the 
multimeric protein to form a reaction complex. 
5. Removing the reaction complex-containing plant cells from the binding 
admixture and thereby separating the metal ion from the fluid sample. 
Chelating agents useful in practicing this method include ethylene diamine 
tetraacetic acid (EDTA) and Bis(bis-carboxy methyl amino propyl) phenyl 
isothiocyanate (CITC). See for example, Meares, et al., Analytical 
Biochemistry, 142:68-78 (1984). The fluid sample may be either a gas or 
liquid sample and, when admixed with a chelating agent, forms a chelating 
admixture. 
The chelating admixture is maintained for a time period sufficient for the 
metal to bind the chelating agent and form a metal ion chelation complex. 
The amount of time required for the metal ion to bind the chelating agent 
will depend upon at least the type of chelating agent employed and the 
concentration of the metal. The metal ion chelation complex is formed when 
at least one metal ion associates with its chelating agent and becomes 
bound to that chelating agent to form a complex. 
This metal ion chelation complex is commingled with plant cells of the 
present invention. These plant cells contain a multimeric protein capable 
of specifically binding the metal ion chelation complex. For example, the 
plant cells may contain an immunoglobulin that is immunospecific for a 
metal chelation complex similar to those immunoglobulin molecules 
described by Reardon, et al., Nature, 316:265-268 (1985) and Meares, et 
al., Analytical Biochemistry, 142:68-78 (1984). 
The binding admixture is maintained for a time period sufficient for the 
metal ion chelation complex to enter the plant cells and bind the 
multimeric protein to form a reaction complex with the plant cells. The 
binding admixture must be maintained under conditions which allow the 
metal ion chelation complex to bind the multimeric protein. Such 
conditions are well known to those skilled in the art. The amount of time 
required for the metal ion chelation complex to enter the plant cell will 
vary and will depend at least upon the concentration and size of the metal 
ion chelation complex. The metal ion chelation complex may enter the plant 
cells passively, for example by diffusion, or may be forced under pressure 
into the plant cells. The reaction complex formed when the metal ion 
chelation complex binds to the multimeric protein present in the plant 
cells consists of the metal ion bound to the chelating agent, the 
chelating agent and the multimeric protein. The reaction 
complex-containing plant cells are then removed from the binding admixture 
thereby separating the metal ion from the fluid sample. The plant cells 
may be removed using the methods well known to those skilled in the art 
and include mechanically removing, filtration, sedimentation and other 
separation means. When the plant cells utilized for this method constitute 
a viable plant, this method concentrates the metal within the plant. 
Transgenic plants of the present invention can be produced from any 
sexually crossable plant species that can be transformed using any method 
known to those skilled in the art. Useful plant species are dicotyledons 
including tobacco, tomato, the legumes, alfalfa, oaks, and maples,; 
monocotyledons including grasses, corn, grains, oats, wheat, and barley; 
and lower plants including gymnosperms, conifers, horsetails, club mosses, 
liver warts, horn warts, mosses, algaes, gametophytes, sporophytes of 
pteridophytes. 
The transgenic plants of the present invention contain polypeptide coding 
genes operatively linked to a promoter. Useful promoters are known to 
those skilled in the art and include inducible promoters, viral promoters, 
synthetic promoters, constitutive promoters, temporally regulated 
promoters, spatially regulated promoters, and spatiotemporally regulated 
promoters. 
In preferred embodiments, the transgenic plants of the present invention 
contain an immunoglobulin product. Useful immunoglobulin products are well 
known to one skilled in the immunoglobulin art and include an 
immunoglobulin heavy chain, an immunoglobulin molecule comprised of a 
heavy and a light chain, one half of an immunoglobulin molecule, a Fab 
fragment, a Fv fragment, and proteins known as single chain antigen 
binding proteins. The structures of immunoglobulin products are well known 
to those skilled in the art and described in Basic and Clinical 
Immunology, by Stites, et al., 4th ed., Lange Medical Publications, Los 
Altos, Calif. The structure of single chain antigen binding proteins has 
been described by Bird et al., Science, 242:423-426 (1988) and U.S. Pat. 
No. 4,704,692 by Ladner. 
The immunoglobulins, or antibody molecules, are a large family of molecules 
that include several types of molecules, such as IgD, IgG, IgA, IgM and 
IgE. The antibody molecule is typically comprised of two heavy (H) and 
light (L) chains with both a variable (V) and constant (C) region present 
on each chain. Several different regions of an immunoglobulin contain 
conserved sequences useful for isolating the immunoglobulin genes using 
the polymerase chain reaction. Extensive amino acid and nucleic acid 
sequence data displaying exemplary conserved sequences is compiled for 
immunoglobulin molecules by Kabat et al., in Sequences of Proteins of 
Immunological Interest, National Institute of Health, Bethesda, Md. 
(1987). 
The V region of the H or L chain typically comprises four framework (FR) 
regions (FIG. 1) each containing relatively lower degrees of variability 
that includes lengths of conserved sequences. The use of conserved 
sequences from the FR1 and FR4 (J region) framework regions of the V.sub.H 
is a preferred exemplary embodiment and is described herein in the 
Examples. Framework regions are typically conserved across several or all 
immunoglobulin types and thus conserved sequences contained therein are 
particularly suited for isolating the variable types. 
One particularly useful immunoglobulin product is an immunoglobulin heavy 
chain. An immunoglobulin heavy chain consists of an immunoglobulin heavy 
chain variable region and an immunoglobulin constant region. The 
immunoglobulin heavy chain variable region is a polypeptide containing an 
antigen binding site (and antibody combining site). Therefore, the 
immunoglobulin heavy chain variable region is capable of specifically 
binding a particular epitope. Preferably, the V.sub.H will be from about 
110 to about 125 amino acid residues in length. The amino acid residue 
sequence will vary widely, depending on the particular antigen the V.sub.H 
is capable of binding. Usually, there will be at least two cysteines 
separated by about 60-75 amino acid residues that are joined to one 
another by a disulfide bond. 
The immunoglobulin constant region (C.sub.H) can be of the alpha, gamma 1, 
gamma 2, gamma 3, delta, mu, or epsilon human isotypes. If the 
immunoglobulin heavy chain is derived from a mouse the C.sub.H may be of 
the alpha, gamma 1, gamma 2a, gamma 2b, gamma 3, delta, mu, or epsilon 
isotypes. The C.sub.H will be of an isotype that is normally present in 
the animal species that it was isolated from. The C.sub.H may also consist 
of domains derived from different isotypes to enhance or confer a given 
biological function. Genes containing the DNA sequence from several 
different constant region isotypes may be combined to produce a chimeric 
gene that encodes a chimeric C.sub.H polypeptide. The DNA and protein 
sequences are easily obtained from available sources. See for example, 
Early Hood, Genetic Engineering, Setlow and Hollaender, eds., Vol. 3, 
Plenum Publishing Corporation, (1981), pages 157-188; and Kabat et al., 
Sequences of Immunological Interest, National Institutes Of Health, 
Bethesda, Md. (1987). These two sources also contain a number of sequences 
for V.sub.H, V.sub.L and C.sub.L genes and proteins. 
Preferred immunoglobulin products are those that contain an immunoglobulin 
heavy chain described above and an immunoglobulin light chain. 
Immunoglobulin light chains consist of an immunoglobulin light chain 
variable region (V.sub.L) and r an immunoglobulin light chain constant 
region (C.sub.L). The V.sub.L will be from about 95 to about 115 amino 
acid residues in length. One skilled in the art will understand that there 
are two isotypes of C.sub.L that are present in both human and mouse, the 
lambda isotype and the kappa isotype. 
In other preferred embodiments the immunoglobulin product consists of 
V.sub.H alone, or of a V.sub.H associated with a V.sub.L to form a Fv 
fragment. 
The contemplated transgenic plants contain a multimeric protein. This 
multimeric protein may be an immunoglobulin product described above, an 
enzyme, a receptor capable of binding a specific ligand, or an abzyme. 
An enzyme of the present invention is a multimeric protein wherein at least 
two polypeptide chains are present. These two polypeptide chains are 
encoded by genes introduced into the transgenic plant by the method of the 
present invention. Useful enzymes include aspartate transcarbamylase and 
the like. 
In another preferred embodiment of the present invention, a multimeric 
protein is a receptor capable of binding a specific ligand. Typically this 
receptor is made up of at least two polypeptide chains encoded by genes 
introduced into the transgenic plant by a method of the present invention. 
Examples of such receptors and their respective ligands include 
hemoglobin, O.sub.2 ; protein kinases, cAMP; and the like. 
In another preferred embodiment of the present invention the immunoglobulin 
product present is an abzyme constituted by either an immunoglobulin heavy 
chain and its associated variable region, or by an immunoglobulin heavy 
chain and an immunoglobulin light chain associated together to form an 
immunoglobulin molecule, a Fab, Fv or a substantial portion of an 
immunoglobulin molecule. Illustrative abzymes include those described by 
Tramontano et al., Science , 234: 1566-1570 (1986): Pollack et al., 
Science. 234 :1570-1573 (1986): Janda et al., Science, 241:1188-1191 
(1988); and Janda et at., Science, 244: 437-440 (1989). 
Typically a multimeric protein of the present invention contains at least 
two polypeptides; however, more than two peptides can also be present. 
Each of these polypeptides is encoded by a separate polypeptide coding 
gene. The polypeptides are associated with one another to form a 
multimeric protein by disulfide bridges, by hydrogen bonding, or like 
mechanisms. 
Included as part of the present invention are transgenic plants that are 
produced from or are the progeny of a transgenic plant of the present 
invention. These transgenic plants contain the same multimeric protein as 
that contained in the parental transgenic plant. Such plants may be 
generated either by asexually propagating the parental plant or by 
self-pollination. The process of asexually propagating and 
self-pollinating a plant are well known. 
In a further aspect, the present invention contemplates a transgenic plant 
that contains a complex. Generally, such a complex-containing transgenic 
plant is obtained by adding a chelating agent to a fluid sample to form a 
chelating admixture, maintaining the admixture for a time period 
sufficient for any metal present in the fluid sample to bind the chelating 
agent and form a metal chelation complex, commingling the metal chelation 
complex with transgenic plant cells of the present invention to form a 
binding admixture, and maintaining the binding admixture for a time period 
sufficient for the metal chelation complex to enter the plant cells and 
bind the multimeric protein present in the plant cells to form a complex 
within the plant cells. 
Also contemplated by the present invention are transgenic plants containing 
a reaction complex consisting of a metal chelation complex and an 
immunoglobulin product. Typically, this transgenic plant will be produced 
by a method the present invention. 
D. Biologically Active Glycopolypeptide Multimers 
The present invention contemplates a biologically active glycopolypeptide 
multimer comprising at least two polypeptides, one of the polypeptides 
having (a) an immunoglobulin amino acid residue sequence, and (b) an 
oligosaccharide comprising a core portion and 
N-acetylglucosamine-containing outer branches, such that the multimer is 
free from sialic acid residues. 
In preferred embodiments, the biologically active glycopolypeptide multimer 
includes an amino acid residue sequence of an immunoglobulin superfamily 
molecule, such as an amino acid residue sequence of an immunoglobulin, a 
molecule of the T cell receptor complex, a major histocompatibility 
complex antigen and the like. Particularly preferred are biologically 
active glycopolypeptide multimers that contain an amino acid residue 
sequence of an immunoglobulin heavy chain, an immunoglobulin heavy chain 
variable region or a portion of an immunoglobulin heavy chain variable 
region. Glycopolypeptide multimers having an amino acid residue sequence 
of an immunoglobulin light chain, and immunoglobulin light chain variable 
region and portions of an immunoglobulin light chain variable region are 
also preferred. 
In a preferred embodiment, the biologically active glycopolypeptide 
multimer comprises a polypeptide having a glycosylated core portion as 
well as N-acetylglucosamine containing outer branches and an amino acid 
residue sequence of an immunoglobulin molecule that is bonded to at least 
one other polypeptide including another amino acid residue sequence. In 
preferred embodiments, the other polypeptide may include an amino acid 
residue sequence of an immunoglobulin superfamily molecule, an 
immunoglobulin molecule, an immunoglobulin heavy chain, an immunoglobulin 
heavy chain variable region, a portion of an immunoglobulin heavy chain 
variable region, an immunoglobulin light chain, an immunoglobulin light 
chain variable region, or a portion of an immunoglobulin light chain 
region. 
In other preferred embodiments, the glycopolypeptide multimer further 
comprises immunoglobulin J chain bonded to the immunoglobulin molecule or 
a portion of the immunoglobulin molecule present in the glycopolypeptide 
multimer. J chain is a polypeptide that is associated with polymeric IgA 
and IgM and other immunoglobulins such as IgG, IgD, IgE, and the other 
various subclasses of these immunoglobulin isotypes. 
The amino acid composition of both human and mouse J chain has been 
described by Mole et al., Biochemistry, 16:3507 (1977), Max and Korsmeyer, 
J. Exp. Med., 161:832 (1985), Cann et al., Proc. Natl. Acad. Sci., USA. 
79:6656 (1982), and Koshland, Annu. Rev. Immunol., 3:425 (1985). J chain 
has 137 amino acid residues with a high proportion of acidic amino acids, 
low numbers of glycine, threonine, cysteine, and only one methionine. The 
J chain contains 8 cysteine residues, 6 of which are involved in the 
formation of intrachain disulfide bonds and 2 are connected to the 
penultimate cysteine residues of the immunoglobulin heavy chain such as 
the alpha or mu heavy chain as described by Mendez et al., Biochem. 
Biophys. Res. Commun., 55:1291 (1973), Mesteckey et al., Proc. Natl. Acad. 
Sci., USA. 71:544 (1974), Mesteckey and Schrohenloher, Nature, 249:650 
(1974). 
In preferred embodiments, the glycopolypeptide multimer also comprises a 
secretory component bonded to the Fc region of the immunoglobulin heavy 
chain amino acid residue sequence present in the glycopolypeptide 
multimer. Secretory component is comprised of a single polypeptide chain 
with 549 to 558 amino acid residues and large amounts of carbohydrates 
attached by N-glycosidic bonds to asparagine residues as 5-7 
oligosaccharide side chains. See, Mostov et al., Nature, 308:37 (1984); 
Eiffert et al., Hoppe Seyler's C. Physiol. Chem., 365:1489 (1984); 
Heremans, N The Antioens, M. Sela ed., 2:365, Academic Press New York 
(1974); Tomana et al., Ana. Biochem., 89:110 (1978); Purkayasthaa et al., 
J. Biol. Chem., 254:6583 (1979); and Mizoguchi et al., J. Biol. Chem., 
257:9612 (1982). Secretory component contains 20 cysteine residues that 
are involved in intrachain disulfide bonding. In preferred embodiments, 
secretory component is disulfide bonded to a cysteine residue present in 
the Fc region of the immunoglobulin heavy chain present in the 
glycopolypeptide multimer. 
The present invention contemplates a glycopolypeptide multimer comprising a 
polypeptide having a glycosylated core portion as well as 
N-acetylglucosamine containing outer branches. The multimer is free from 
detectable sialic acid residues. The polypeptide has a glycosylated core 
portion including an N-acetylglucosamine oligosaccharide bonded via its 
C(1) carbon directly to the amide group of an asparagine amino acid 
residue present in the polypeptide. The glycosylated core portion has the 
structure Man.alpha.1-3(Man.alpha.1-6) Man.beta.11-4GlcNAc.beta.1-4 
GlcNAc-Asn contained within the boxed area in FIG. 6. The polypeptide also 
has outer oligosaccharide branches (outer branches) that contain 
N-acetylglucosamine. Both complex and hybrid asparagine-linked 
oligosaccharides contain N-acetylglucosamine containing outer branches, 
while high mannose oligosaccharides do not. Bacterial cells do not include 
glycosylated core portions attached to asparagine amino acids. Yeast cells 
do not have asparagine-linked oligosaccharides of either the complex or 
hybrid type and therefore yeast do not have N-acetylglucosamine containing 
outer branches. Plant cells are capable of producing a polypeptide having 
a glycosylated core portion linked to an asparagine amino acid as well as 
N-acetylglucosamine containing outer branches. 
The glycopolypeptide multimer comprises a polypeptide that has a 
glycosylated core portion as well as N-acetylglucosamine containing outer 
branches. The entire the multimer is free from detectable sialic acid 
residues. Sialic acid, the predominant terminal carbohydrate of mammalian 
glycoproteins, has not been identified as a carbohydrate residue of plant 
proteins. The terminal carbohydrate residues found in plants include 
xylose, fucose, N-acetylglucosamine, mannose or galactose as has been 
described by Sturm et al., J. Biol. Chem.. 262:13392 (1987). In other 
respects, plant glycoproteins and carbohydrates attached to those proteins 
are very similar to mammalian glycoproteins. A glycopolypeptide multimer 
produced in a plant comprises a polypeptide having a glycosylated core 
portion as well as N-acetylglucosamine containing outer branches but is 
free from detectable sialic acid residues. 
A gene coding for a polypeptide having within its amino acid residue 
sequence, the N-linked glycosylation signal, 
asparagine-X-serine/threonine, where X can be any amino acid residue 
except possibly proline or aspartic acid, when introduced into a plant 
cell would be glycosylated via oligosaccharides linked to the asparagine 
residue of the sequence (N-linked). See, Marshall, Ann. Rev. Biochem., 
41:673 (1972) and Marshall, Biochem. Soc. Symp., 40:17 (1974) for a 
general review of the polypeptide sequences that function as glycosylation 
signals. These signals are recognized in both mammalian and in plant 
cells. However in plant cells these signals do not result in 
asparagine-linked oligosaccharides that contain terminal sialic acid 
residues as are found in mammalian cells when expressed in a plant cell, a 
polypeptide containing the N-linked glycosylation signal sequence would be 
glycosylated to contain a glycosylated core portion as well as 
N-acetylglucosamine containing outer branches and would be free from 
detectable sialic acid residues. 
A glycopolypeptide multimer, a protein, or a polypeptide of the present 
invention is free from detectable sialic acid residues as evidenced by its 
lack of specific binding to lectins specific for sialic acid such as wheat 
germ agglutinin or Ricinus communis, agglutinin. Methods for determining 
the binding of a glycosylated polypeptide chain to a particular lectin are 
well known in the art. See, e.g., Faye et al., Ana. Biochem., 149:218 
(1985) and Goldstein et al., Adv. Carbohydr. Chem. Biochem., 35:127 
(1978). Typical methods for determining whether a glycosylated polypeptide 
chain binds to a particular lectin include methods using lectin columns, 
and methods where the glycosylated polypeptide is bound to nitrocellulose 
and probed with a biotinylated lectin. The exact specificity of the lectin 
may be determined by competing the lectin binding with a particular 
oligosaccharide such as a sialic acid residue. The common sialic acid 
residues are shown in FIG. 7. 
Immunoglobulin superfamily molecules, and immunoglobulins may have various 
carbohydrate groups attached to them. Typically the carbohydrate is found 
on the immunoglobulin heavy chain constant region except for a few 
instances when the tripeptide acceptor sequence 
asparagine-X-serine/threonine(N-linked signal), is found within the heavy 
chain variable region. Other immunoglobulin superfamily molecules 
containing the tripeptide acceptor sequence (N-linked glycosylation 
sequence) within its amino acid residue sequence would also contain 
carbohydrate groups attached to the asparagine of that tripeptide acceptor 
sequence. FIG. 8 shows the typical carbohydrate groups attached to 7 human 
heavy chains as described by Jeske and Capra, in Fundamental Immunology, 
W. E. Paul, ed., Raven Press, New York, N.Y. (1984). The carbohydrate 
attachment sites are highly conserved between various species and the 
comparable classes of immunoglobulin heavy chains. Table B shows the 
various oligosaccharides on each of the human immunoglobulin heavy chains. 
TABLE B 
__________________________________________________________________________ 
Structural Characteristics of Human Immunoglobulin Heavy Chains 
Constant Region 
No. of residues 
Whole Chain Interchain 
Position of 
Oligosacchrides 
(approximate) 
Chain Domains 
bridges 
H-L bridge 
GlcN 
GalN 
Hinge 
C Region 
__________________________________________________________________________ 
gamma 1 4 3 220 1 0 15 330 
gamma 2 4 5 131 1 0 12 325 
gamma 3 4 12 131 1 0 62 375 
gamma 4 4 3 131 1 0 14 325 
alpha 1 4 5 133 2 5 26 350 
alpha 2 A2m(1) 
4 4 missing 
4 0 13 340 
alpha 2 A2m(2) 
4 5 133 5 0 13 340 
mu 5 4 140 5 0 0 450 
epsilon 5 3 127 6 0 0 420 
delta 4 2 128 3 4 or 5 
64 380 
__________________________________________________________________________ 
Preferably, the polypeptide present in the glycopolypeptide multimer 
includes the N-linked glycosylation signal within the immunoglobulin 
molecule amino acid residue sequence. In other preferred embodiments, the 
N-linked glycosylation is present in the region of the polypeptide that is 
not an immunoglobulin residue sequence. 
In preferred embodiments, the biologically active glycopolypeptide multimer 
comprises secretory IgA. Secretory IgA is made up of four immunoglobulin 
alpha heavy chains, four immunoglobulin light chains, J chain and 
secretory component all bonded together to form a secretory IgA molecule 
containing an IgA dimer. The secretory IgA molecule contains heavy and 
light chain variable regions that bind specifically to an antigen. The 
secretory IgA molecule may contain either IgA.sub.1 or IgA.sub.2 
molecules. For a general discussion of secretory IgA, see Mesteckey et 
al., Advances in Immunology, 40:153 (1987). 
The final assembled secretory IgA of animals is the product of two distinct 
cell types: plasma cells that produce IgA with attached J chain and 
epithelial cells that produce secretory IgA. The transytosis and secretion 
of the complex is the result of the membrane only at the luminal surface 
of the cell. The interaction of the four components of the complex (alpha, 
gamma, J, SC) results in an immunoglobulin structure which is 
exceptionally resistant to the degradative environment associated with 
mucosal surfaces. 
In other preferred embodiments the biologically active glycopolypeptide 
multimer is a secretory IgM molecule that contains five IgM molecules, 
three J chain molecules and secretory component all disulfide bonded 
together. 
Both secretory immunoglobulins (IgM and IgA) are resistant to proteolysis 
and degradation and therefore are active when present on mucosal surfaces 
such as the lungs or the gastrointestinal tract. See, Tomasi, N. Basic and 
Clinical Immunology, p. 198, Lange Medical Publications, Los Altos, Calif. 
(1982). 
In preferred embodiments, a biologically active glycopolypeptide multimer 
has within it at least one catalytic site. This catalytic site may be an 
enzymatic site that is formed by one or more polypeptides. The catalytic 
site present is typically defined by an amino acid residue sequence that 
is known to form a catalytic site alone or together with the amino acid 
residue sequences of other polypeptides. This catalytic site may be the 
active site of an enzyme, or the binding site of an immunoglobulin. See, 
e.g., Tramontano et al., Science, 234:1566 (1986). The present invention 
also contemplates other enzymes containing a catalytic site such as the 
enzymes described in Biochemistry Worth Publishers, Inc., New York (1975). 
In other preferred embodiments, the present invention contemplates a 
biologically active glycopolypeptide multimer comprising a polypeptide 
having a glycosylated core portion as well as N-acetylglucosamine 
containing outer branches and includes an immunoglobulin molecule amino 
acid residue sequence, bonded to another polypeptide including a different 
immunoglobulin molecule amino acid residue sequence where the multimer is 
free from detectable sialic acid. 
Catalytic glycopolypeptide multimers are contemplated wherein the catalytic 
site of the glycopolypeptide multimer is comprised of a first and second 
portion. The first portion of the catalytic site is also defined by an 
immunoglobulin amino acid residue sequence. The second portion of the 
catalytic site is defined by a different immunoglobulin amino acid residue 
sequence. The first and second portions of the catalytic site are 
associated together to form a greater portion of the catalytic site. In 
more preferred embodiments, the first portion of the catalytic site is 
defined by an immunoglobulin heavy chain variable region amino acid 
residue sequence and the second portion of the catalytic site is defined 
by an immunoglobulin light chain variable region amino acid residue 
sequence that is associated with the heavy chain amino acid residue 
sequence to form a larger portion at the catalytic site. 
The present invention also contemplates a biologically active 
glycopolypeptide multimer comprising: 
(i) A polypeptide having a glycosylated core portion as well as a 
N-acetylglucosamine-containing outer branches and an immunoglobulin 
molecule amino acid residue sequence and the polypeptide does not bind to 
a mouse immunoglobulin binding lectin; and 
(ii) another polypeptide containing a different immunoglobulin molecule 
amino acid residue sequence, where this another polypeptide is bonded to 
the polypeptide. 
Mouse immunoglobulin binding lectins include lectins that specifically bind 
terminal sialic acid residues such as wheat germ agglutinin and Ricinus 
communis agglutinin. A mouse immunoglobulin binding lectin is specific for 
terminal sialic acid residues and thus does not bind an immunoglobulin 
produced in a plant cell because immunoglobulins produced in plants do not 
contain terminal sialic acid residues. See, Osawa et al., Ana. Rev. 
Biochem., 56:21-42 (1987) for a general discussion of lectin binding 
properties. 
E. Passive Immunizations Using Immunoglobulins Produced in Plants 
Methods of passively immunizing an animal against a preselected ligand by 
contacting a composition comprising a biologically active glycopolypeptide 
multimer of the present invention that is capable of binding a preselected 
ligand with a mucosal surface of an animal are contemplated by the present 
invention. 
Biologically active glycopolypeptide multimers such as immunoglobulin 
molecules capable of binding a preselected antigen can be efficiently and 
economically produced in plant cells. These immunoglobulin molecules do 
not contain sialic acid yet do contain core glycosylated portions and 
N-acetylglucosamine containing outer branches. In preferred embodiments, 
the immunoglobulin molecule is either IgA, IgM, secretory IgM or secretory 
IgA. 
Secretory immunoglobulins, such as secretory IgM and secretory IgA are 
resistant to proteolysis and denaturation and therefore are desirable for 
use in harsh environments. Contemplated harsh environments include acidic 
environments, protease containing environments, high temperature 
environments, and other harsh environments. For example, the 
gastrointestinal tract of an animal is a harsh environment where both 
proteases and acid are present. See, Kobayishi et al., Immunochemistry, 
10:73 (1973). Passive immunization of the animal is produced by contacting 
the glycopolypeptide multimer with a mucosal surface of the animal. 
Animals contain various mucosal surfaces including the lungs, the 
digestive tract, the nasopharyngeal cavity, the urogenital system, and the 
like. Typically, these mucosal surfaces contain cells that produce various 
secretions including saliva, lacrimal fluid, nasal fluid, tracheobronchial 
fluid, intestinal fluid, bile, cervical fluid, and the like. 
In preferred embodiments the glycopolypeptide multimer, such as the 
immunoglobulin molecule is immunospecific for a preselected antigen. 
Typically, this antigen is present on a pathogen that causes a disease 
that is associated with the mucosal surface such as necrotizing 
enterocolitis, diarrheal disease, and cancer caused by carcinogen 
absorption in the intestine. See e.g., McNabb and Tomasi, Ann. Rev. 
Microbiol., 35:477 (1981) and Lawrence et al., Science, 243:1462 (1989). 
Typical pathogens that cause diseases associated with a mucosal surface 
include both bacterial and viral pathogens such as E. coli, S. 
typhimurium, V. cholera. and S. mutans. The glycopolypeptide multimer is 
capable of binding to these pathogens and preventing them from causing 
mucosal associated or mucosal penetrating diseases. 
In preferred embodiments, the composition contacted with the animal mucosal 
surface comprises a plant material and a biologically active 
glycopolypeptide multimer that is capable of binding a preselected ligand. 
The plant material present may be plant cell walls, plant organelles, 
plant cytoplasm, intact plant cells containing the glycopolypeptide 
multimer, viable plants, and the like. This plant cell material is present 
in a ratio from about 10,000 grams of plant material to about 100 
nanograms of glycopolypeptide multimer, to about 100 nanograms of plant 
material for each 10 grams of glycopolypeptide multimer present. In more 
preferred embodiments, the plant material is present in a ratio from about 
10,000 grams of plant material for each 1 mg of glycopolypeptide multimer 
present, to about a ratio of 100 nanograms of plant material present for 
each gram of glycopolypeptide multimer present. In other preferred 
embodiments, the plant material is present in a ratio from about 10,000 
grams of plant material for each milligram of glycopolypeptide multimer 
present to about 1 mg of plant material present for each 500 mg of 
glycopolypeptide multimer present. 
In preferred embodiments, the composition comprising the biologically 
active glycopolypeptide multimer is a therapeutic composition. The 
preparation of therapeutic compositions which contain polypeptides or 
proteins as active ingredients is well understood in the art. Therapeutic 
compositions may be liquid solutions or suspensions, solid forms suitable 
for solution in, or suspension in a liquid prior to ingestion may also be 
prepared. The therapeutic may also be emulsified. The active therapeutic 
ingredient is typically mixed with inorganic and/or organic carriers which 
are pharmaceutically acceptable and compatible with the active ingredient. 
The carriers are typically physiologically acceptable excipients 
comprising more or less inert substances when added to the therapeutic 
composition to confer suitable consistencies and form to the composition. 
Suitable carriers are for example, water, saline, dextrose, glycerol, and 
the like and combinations thereof. In addition, if desired the composition 
can contain minor amounts of auxiliary substances such as wetting or 
emulsifying agents and pH buffering agents which enhance the effectiveness 
of the active ingredient. Therapeutic compositions containing carriers 
that have nutritional value are also contemplated. 
In preferred embodiments, a composition containing a biologically active 
glycopolypeptide multimer comprises an immunoglobulin molecule that is 
immunospecific for a pathogen antigen. Pathogens are any organism that 
causes a disease in another organism. Particularly preferred are 
immunoglobulins that are immunospecific for a mucosal pathogen antigen. A 
mucosal pathogen antigen is present on a pathogen that invades an organism 
through mucosal tissue or causes mucosal associated diseases. Mucosal 
pathogens include lung pathogens, nasal pathogens, intestinal pathogens, 
dental pathogens, and the like. For a general discussion of pathogens, 
including mucosal pathogens, see, Davis et al., Microbiology, 3rd ed., 
Harper and Row, Hagerstown, Md. (1980). 
Antibodies immunospecific for a pathogen may be produced using standard 
monoclonal antibody production techniques. See, Antibodies: A Laboratory 
Manual, Harlow et al., eds., Cold Spring Harbor, N.Y. (1988). The genes 
coding for the light chain and heavy chain variable regions can then be 
isolated using the polymerase chain reaction and appropriately selected 
primers. See, Orlandi er al., Proc. Natl. Acad. Sci., U.S.A.. 86:3833 
(1989) and Huse et al., Science, 246:1275 (1989). The variable regions are 
then inserted into plant expression vectors, such as the expression 
vectors described by Hiatt et al., Nature, 342:76-78 (1989). 
In a preferred embodiment, the biologically active glycopolypeptide 
multimer is a immunoglobulin immunospecific for an intestinal pathogen 
antigen. Particularly preferred are immunoglobulins immunospecific for 
intestinal pathogens such as bacteria, viruses, and parasites that cause 
disease in the gastrointestinal tract, such as E. coli, Salmonellae, 
Vibrio cholerae, Salmonellae typhimurium, and Streotococcus mutans. Also 
contemplated by the present invention are glycopolypeptide multimers that 
are immunoglobulins immunospecific for Diphtheria toxin, such as the 
antibody produced by the hybridoma ATCC No. HB 8329; antibodies 
immunospecific Pseudomonas aeruginosa exotoxin A, such as the antibody 
produced by the hybridoma D253-15-6 (ATCC No. H 8789); immunoglobulins 
immunospecific for Ricin A or B chain, such as the immunoglobulins 
produced by hybridomas TFT A1 (ATCC No. CRL 1771) or TFTBl (ATCC No. 
1759); immunoglobulins immunospecific for Schistosoma mansoni 
glycoprotein, such as the antibody produced by hybridoma 130 C/2B/8 (ATCC 
No. 8088); immunoglobulins immunospecific for Shigella SHIGA toxin and 
Shigella-like toxin, such as the antibodies produced by hybridoma 13C4 
(ATCC No. 1794); immunoglobulins immunospecific for tetanus toxoid, such 
as the immunoglobulins produced by hybridomas 9F12 (ATCC No. HB8177) and 
hybridoma SA13 (ATCC No. HB8501); immunoglobulins immunospecific for 
Trichinella spiralis, such as hybridoma 7C.sub.2 C.sub.5 C.sub.12 (ATCC 
No. HB 8678); immunoglobulins immunospecific for Dengue viruses or 
complexes, such as the immunoglobulins produced by D3-2H2-9-21 (ATCC No. 
HB 114), hybridoma 15F3-1 (ATCC No. HB 47), hybridoma 3H51 (ATCC No. HB 
46), hybridoma 5D4-11 (ATCC No. HB 49), hybridoma IH10-6 (ATCC No. HB 48); 
immunoglobulins immunospecific for Hepatitis B surface antigen, such as 
hybridoma H25B10 (ATCC No. CRL 8017), hybridoma H21 F8-1 (ATCC No. CRL 
8018 ; immunoglobulins immunospecific for Herpes simplex viruses, such as 
the immunoglobulin produced by hybridoma 1D4 (ATCC No. HB 8068), hybridoma 
39-S (ATCC No. HB 8180), hybridoma 52-S (ATCC No. HB 8181), hybridoma 3NI 
(ATCC No. HB 8067); immunoglobulins immunospecific for influenza virus, 
such as the immunoglobulins produced by HK-PEG-1 (ATCC No. CL 189), 
hybridoma M2-lC6-4R3 (ATCC No. HB64); immunoglobulins immunospecific for 
parainfluenza virus, such as the immunoglobulin produced by hybridoma 
9-3-4 (ATCC No. 8935); and immunoglobulins immunospecific for 
parvoviruses, such as the immunoglobulin produced by 3C9-D11-H11 (ATCC No. 
CRL 1745). 
In other preferred embodiments, the qlycopolypeptide multimer present in 
the composition is an immunoglobulin molecule that is immunospecific for a 
dental pathogen antigen such as Streptococcus mutans and the like. 
Particularly preferred are immunoglobulins immunospecific for a 
Streotococcus mutans antigen such as the immunoglobulin produced by 
hybridoma 15B2 (ATCC No. HB 8510). 
The present invention contemplates producing passive immunity in an animal, 
such as a vertebrate. In preferred embodiments, passive immunity is 
produced in fish, birds, reptiles, amphibians, or insects. In other 
preferred embodiments passive is produced in a mammal, such as a human, a 
domestic animal, such as a ruminant, a cow, a pig, a horse, a dog, a cat, 
and the like. In particularly preferred embodiments, passive immunity is 
produced in an adult mammal. 
In preferred embodiments, passive immunity is produced in an animal, such 
as a mammal that is weaned and therefore no longer nurses to obtain milk 
from its mother. Passive immunity is produced in such an animal by 
administering to the animal a sufficient amount of a composition 
containing a glycopolypeptide multimer immunospecific for a preselected 
ligand to produce a prophylactic concentration of the glycopolypeptide 
multimer within the animal. A prophylactic concentration of a 
glycopolypeptide multimer, such as an immunoglobulin is an amount 
sufficient to bind to a pathogen present and prevent that pathogen from 
causing detectable disease within the animal. The amount of composition 
containing the glycopolypeptide multimer required to produce a 
prophylactic concentrations will vary as is well known in the art with the 
size of the animal, the amount of pathogen present, the affinity of the 
particular glycopolypeptide multimer for the pathogen, the efficiency with 
which the particular glycopolypeptide multimer is delivered to its active 
location within the animal, and the like. 
The present invention also contemplates a method for providing passive 
immunity against a pathogen to an animal, by administering to the animal 
an encapsulated, biologically active glycopolypeptide multimer capable of 
binding a pathogen antigen in an amount sufficient to establish within the 
animal a prophylactic concentration of the multimer that contains a 
polypeptide having a glycosylated core portion as well as 
N-acetylglucosamine containing outer branches and an amino acid residue 
sequence of an immunoglobulin molecule and where the multimer is free from 
detectable sialic acid residues. 
In preferred embodiments, the biologically active glycopolypeptide multimer 
is encapsulated in a protective coating. The encapsulation material may be 
a membrane, a gel, a polymer or the like. The encapsulation material 
functions to protect the material it contains and to control the flow of 
material in and out of the encapsulation device. In preferred embodiments, 
the glycopolypeptide multimer is encapsulated within a plant cell wall, a 
plant cell, a micelle, an enteric coating, and the like. 
In preferred embodiments, glycopolypeptide multimers, such as, tissue 
plasminogen activator, recombinant human insulin, recombinant alpha 
interferon and growth hormone, have been successfully administered and are 
therapeutically effective through buccal, nasal, and rectal mucosa using 
various approaches. Eppstein et al., Alternative Delivery Systems for 
Peptides and Proteins as Drugs, CRC Critical. Rev. in Therapeutic Drug 
Carrier Systems, 5:99-139 (1988). 
In preferred embodiments, the biologically active glycopolypeptide multimer 
is administered by intranasal formulations in solution. The formulation is 
administered by one of three ways: a single dose through a catheter; 
multiple doses through metered dose pumps (also called nebulizers); and 
multiple doses through the use of metered dose pressurized aerosols. If 
desired, the absorption of the peptide or protein across the nasal mucosa, 
may be promoted by adding absorption enhancers including nonionic 
polyoxyethylene ethers, bile salts such as sodium glycocholate (SGC) and 
deoxycholate (DOC), and derivative of fusidic acid such as sodium 
taurodihydrofusidate (STDHF). 
Nasal insulin formulations containing 0.9% weight per volume of sodium 
chloride and 1% DOC, 0.5 U/kg of insulin administered as a spray using a 
metered dose spray pump resulted in rapid elevations of serum insulin. 
Moses et al., Diabetes 32:1040 (1983). Dosages of biologically active 
glycopolypeptide multimers can range from 0.15 mg/kg up to 600 mg/kg, 
preferred dosages range from 0.15 mg/ml up to 200 mg/kg, and most 
preferred dosages range from 1 mg/kg up to 200 mg/kg in a nasal spray 
formulation. In preferred embodiments, the multimer does not cross the 
mucosal membrane and thus absorption enhancers are not required. Several 
dosage forms are available for the rectal delivery of biologically active 
glycopolypeptide multimers. These include suppositories (emulsion and 
suspension types), rectal gelatin capsules (solutions and suspensions), 
and enemas macro: 100 milliliters (ml) or more; and micro: 1 to 20 ml). 
Osmotic pumps designed to deliver a volume of 2 ml in a 24 to 40 hour 
period have also been developed for rectal delivery. Absorption enhancers 
described for nasal formulations are included in the formulations if 
increased transport across rectal mucosa is desired. A preferred 
formulation for rectal administration of the biologically active 
glycopolypeptide multimer consists of the preferred ranges listed above in 
any one of the acceptable dosage forms. 
Biologically active glycopolypeptide multimers can be administered in a 
liposome (micelle) formulation which can be administered by application to 
mucous membranes of body cavities. Juliano et al., J. Pharmacol. Exp. 
Ther., 214:381 (1980). Liposomes are prepared by a variety of techniques 
well known to those skilled in the art to yield several different physical 
structures, ranging from the smallest unilamellar vesicles of 
approximately 20 to 50 nanometers in diameter up to multilamellar vesicles 
of tens of microns in diameter. Gregoriadias, Ed., Liposome Technology, 
1:Crc Press (1984). The biologically active glycopolypeptide multimers in 
the preferred dosages listed for nasal formulations are hydrated with a 
lyophilized powder of multilamellar vesicles to form glycopolypeptide 
containing-liposomes. 
In a more preferred embodiment, biologically active glycopolypeptide 
multimers in the above mentioned preferred dosages are orally administered 
in gelatin capsules which are coated with a azoaromatic cross-linked 
polymer. The azopolymer-coated glycopolypeptide is protected from 
digestion in the stomach and the small intestine. When the 
azopolymer-coated glycopolypeptide reaches the large intestine, the 
indigenous microflora reduce the azo bonds, break the cross-links, and 
degrade the polymer film. This results in the release of the 
glycopolypeptide multimers into the lumen of the colon for subsequent 
local action or absorption. 
Preferably, the pathogen specific glycopolypeptide multimer is administered 
in an amount sufficient to establish a prophylactic concentration of the 
multimer at a particular location in the animal. The amount of multimer 
that is administered to produce a particular prophylactic concentration 
will vary, as is well known in the art, with the amount of pathogen 
present, the exact location in the animal desired to be immunized, the 
affinity of the multimer for the pathogen, the resistance of the multimer 
to denaturation or degradation, the mode of pathogen inactivation, the 
dosage formulation and the like. 
Preferably, the multimer is administered in 10 g to 100,000 g of plant 
material containing about 0.1 mg to 2,000 mg of multimer in 1 to 4 
separate doses each day. This amount of multimer produces a prophylactic 
concentration of about 0.01 mg/kg of body weight to about 2,000 mg/kg of 
body weight. In preferred embodiments, the prophylactic concentration of 
multimer is from about 0.01 mg/kg of body weight to about 600 mg/kg of 
body weight. In other preferred embodiments, the prophylactic 
concentration is from about 0.01 mg/kg body weight to about 200 mg/kg of 
body weight. 
The present invention contemplates a method for providing passive immunity 
to an animal against a preselected ligand, which method comprises 
administering to the animal biologically active glycopolypeptide multimers 
capable of binding a preselected ligand in an amount sufficient to 
establish within the animal a prophylactic concentration of the multimer. 
The multimer administered comprises a polypeptide having a glycosylated 
core portion as well as N-acetylglucosamine-containing outer branches and 
an amino acid sequence of an immunoglobulin molecule, such that the 
multimer is free from detectable sialic acid residues. 
Particularly preferred, is a method for providing passive immunity to an 
animal against a pathogen, which method comprises administering to the 
animal a biologically active glycopolypeptide multimer capable of binding 
a pathogen in amounts sufficient to establish within the animal a 
prophylactic concentration of the multimer. The multimer administered 
comprises a polypeptide having a glycosylated core portion as well as 
N-acetylglucosamine-containing outer branches and an amino acid residue 
sequence of an immunoglobulin molecule, such that the multimer is free 
from detectable sialic acid residues. 
In preferred embodiments, the multimer is administered as a composition 
constituted by the multimer and a material having nutritional value. A 
material having nutritional value is a substance or compound from which 
the animal can derive calories. Typical materials having nutritional value 
include proteins, carbohydrates, lipids, fats, glycoproteins, glycogen, 
and the like. Particularly preferred are materials having nutritional 
value that are plant materials or animal materials. 
In other preferred embodiments, the multimer is administered as a 
composition constituted by the multimer and a physiologically inert 
material. Physiologically inert materials include solutions such as water 
and carrier compounds. 
In other preferred embodiments, a method of passively immunizing an animal 
against a preselected ligand comprising introducing into the 
gastrointestinal tract of an animal a composition comprising plant cell 
walls and a biologically active glycopolypeptide multimer that is capable 
of binding a preselected antigen; said glycopolypeptide multimer 
comprising at least two polypeptides, one of said polypeptides having (a) 
an immunoglobulin amino acid residue sequence, and (b) an oligosaccharide 
comprising a core portion and a N-acetylglucosamine-containing outer 
branches, said multimer being free from sialic acid residues. 
Other preferred embodiments contemplate a method of passively immunizing an 
animal against a preselected antigen, comprising: 
(1) introducing into the gastrointestinal tract of an animal a composition 
comprising plant cells containing a biologically active glycopolypeptide 
multimer that is capable binding a preselected ligand; said multimer 
comprising at least two polypeptides, one of said polypeptides having (a) 
an immunoglobulin amino acid residue sequence, and (b) an oligosaccharide 
comprising a core portion and a N-acetylglucosamine-containing outer 
branches, such that the multimer is free from sialic acid residues; and 
(2) disrupting the plant cell within the gastrointestinal tract, thereby 
releasing the biologically active glycopolypeptide multimer into the 
gastrointestinal tract, and passively immunizing the animal. 
D. Compositions Containing Glycopolypeptide Multimer 
The present invention also contemplates biologically active compositions 
which comprise an encapsulated glycopolypeptide multimer comprising at 
least two polypeptides, one of said polypeptides having (a) an 
immunoglobulin amino acid residue sequence, and (b) an oligosaccharide 
comprising a core portion and a N-acetylglucosamine-containing outer 
branches, such that the multimer is free from sialic acid residues. 
In preferred embodiments the glycopolypeptide multimer is encapsulated in a 
plant cell, a plant cell wall, an enteric coating, a coating, and the 
like. 
Particularly preferred are compositions containing ratios of about 10,000 
grams of plant material to each 100 nanograms of glycopolypeptide multimer 
present to ratios of about 100 nanograms of plant material for each 10 
grams of glycopolypeptide multimer present in the composition. In more 
preferred embodiments, the plant material is present in a ratio from about 
10,000 grams of plant material for each one milligram of glycopolypeptide 
multimer present, to a ratio of about 100 nanograms of plant material 
present for each gram of glycopolypeptide multimer present. In other 
preferred embodiments, the plant material is present in a ratio from about 
10,000 grams of plant material for each milligram of glycopolypeptide 
multimer present to about one milligram of plant material present for each 
500 milligrams of glycopolypeptide multimer present in the composition. 
In other embodiments, the composition further comprises chlorophyll, 
synergistic compounds, medicines, compounds derived from medicinal plants, 
I0 and various pharmaceuticals and the like. Compounds from a medicinal 
plant may be added to the composition by expressing the glycopolypeptide 
multimer in the medicinal plant and then harvesting the plant. 
The present invention also comtemplates a glycopolypeptide multimer 
produced according to the method comprising: 
(a) introducing into the genome of a first member of the plant species a 
first mammalian gene coding for an autogenously linking monomeric 
polypeptide having a N-linked glycosylation signal that is a constituent 
part of the glycopolypeptide multimer to produce a first transformant; 
(b) introducing into the genome of a second member of the same plant 
species another mammalian gene coding for another autogenously linking 
monomeric polypeptide that is a constituent part of the glycopolypeptide 
multimer to produce a second transformant; 
(c) generating from said first and second transformants a progeny 
population; and 
(d) isolating from said progeny population a transgenic plant species that 
produces the glycopolypeptide multimer. 
Other multimers produced by the methods of this invention are contemplated. 
EXAMPLES 
The following examples are intended to illustrate, but not limit, the scope 
of the invention. 
1. Isolation Of An Immunoglobulin Heavy Chain-Coding Gene And An 
Immunoglobulin Light Chain-Coding Gene From The Hybridoma Cell Line 6D4 
Hybridoma cells secreting the 6D4 antibody described by Tramontano et al., 
Science, 234:1566-1570 (1986) were grown to log phase in DMEM medium 
supplemented with 10% fetal calf serum. Total RNA was prepared from 2 
liters of log phase 6D4 hybridoma cells using the methods described by 
Ullrich et al., Science, 196:1313 (1977). Briefly, the 6D4 cells were 
collected by centrifugation and homogenized at room temperature for 20 
seconds in 70 ml of 4M guanidinium thiocyanate containing 5 mM sodium 
citrate at pH 7.0, 0.1M 2-mercaptoethanol (2Me) and 0.5% sodium lauryl 
sarcosinate using a Polytron homogenizer. The homogenate was centrifuged 
briefly for 5 minutes at 8,000 .times.g to remove the insoluble debris. 
About 28 ml of homogenate was layered onto a 10 ml pad of 5.7M CsCl 
(Bethesda Research Laboratories, Gaithersburg, Md.) in 4 mM ethylene 
diamine tetraacetic acid (EDTA) at pH 7.5 in a Beckman SW70 Ti rotor. The 
solution was centrifuged for at least 5 hours at 50,000 revolutions per 
minute (rpm) at 15 C. The supernatant was carefully aspirated and the 
walls of the tubes dried to remove any remaining homogenate. The RNA 
pellet was dissolved in a solution containing 10 mM Tris-HCl at pH 7.4, 2 
mM EDTA and 0.5% sodium dodecyl sulfate (SDS). This solution was extracted 
twice with a phenol solution. The resulting aqueous phase was reextracted 
with solution containing Phenol:chloroform:isoamyl alcohol (25:25:1 by 
volume). The RNA was recovered from the resulting aqueous phase by adding 
110 volume of 3 M sodium acetate and 2 volumes of ethanol. This solution 
was maintained at -20 C. for 12 to 18 hours to precipitate the RNA. The 
solution containing the precipitated RNA was centrifuged for 20 minutes at 
10,000 .times.g at 4 C. to produce a RNA containing pellet. The excess 
salt was removed from the RNA pellet by admixing 5 ml of 70% ethanol to 
the RNA pellet and the solution was centrifuqed for 10 minutes at 10,000 
.times.g at 4 C. The final RNA pellet was dissolved in 0.5 ml of 
DEPC-H.sub.2 O and stored at -70 C. after removing a small aliquot to 
determine the RNA concentration by absorbance at 260 nm. 
Messenger RNA (mRNA) enriched for sequences containing long poly A tracts 
was prepared from the total cellular RNA using the methods described in 
Molecular Cloning: A Laboratory Manual, Maniatis et al., eds., Cold Spring 
Harbor Laboratory, New York (1982). Briefly, the total RNA prepared above 
was resuspended in one ml of DEPC-H.sub.2 O and maintained at 65 C. for 
five minutes. One ml of 2.times. high salt loading buffer consisting of 
100 mM Tris-Cl, 1M sodium chloride (NaCl), 2.0 mM EDTA at pH 7.5, and 1.0% 
sodium dodecyl sulfate (SDS) was added to the resuspended RNA and the 
mixture allowed to cool to room temperature. The mixture was then applied 
to an oligo-dT (Collaborative Research Type 2 or Type 3) column that had 
been previously prepared by washing the oligo-dT with a solution 
containing 0.1M sodium hydroxide and 5 mM EDTA and then equilibrating the 
column with DEPC-H.sub.2 O. The eluate was collected in a sterile 
polypropylene tube and reapplied to the same column after heating the 
eluate for 5 minutes at 65 C. The oligo dT column was then washed with 20 
ml of high salt loading buffer consisting of 50 mM Tris-Cl at pH 7.5, 500 
mM NaCl, 1 mM EDTA at pH 7.5 and 0.5% SDS. The messenger RNA was eluted 
from the oligo dT column with 1 ml of buffer consisting of 10 mM Tris-Cl 
at pH 7.5, 1 mM EDTA at pH 7.5 and 0.05% SDS. The messenger RNA was 
concentrated by ethanol precipitation and resuspended in DEPC H.sub.2 O. 
Complementary DNA (cDNA) was prepared from the mRNA prepared above. The 
first strand synthesis, second strand synthesis, and the fill-in reactions 
were carried out according to the procedures described by Watson et al., 
DNA Cloning Volume I, D. M. Glover, ed., (). Briefly, a solution 
containing 10 ug of mRNA was maintained at 65C for 5 minutes and then 
quickly chilled on ice. The first cDNA strand was synthesized by admixing 
to this solution 100 ul of a reaction mixture containing 50 mM Tris-Cl at 
pH 8.3, 8 mM MgCl.sub.2, 50 mM KCl, 2 ug oligo (dT) 1 mM dATP, 1 mM dGTP, 
1 mM dTTP, 1 mM dCTP, 10 mM DTT, 60 units of RNasin (Promega Corporation, 
Madison, Wis.), 4 ug Actinomycin, 135 units of AMV reverse transcriptase 
and 10 uCi .alpha..sup.32 P-dCTP. This reaction mixture was maintained at 
44C for 1 hour. An additional 60 units of RNasin and 80 units of reverse 
transcriptase were added and the reaction mixture maintained at 44 C. for 
30 minutes. The first strand cDNA synthesis reaction was terminated by 
adding 0.005 ml of a solution containing 50 mM EDTA and 10% SDS. The 
nucleic acids were purified by phenol extraction and then concentrated by 
ethanol precipitation. 
The second strand cDNA was synthesized by admixing all of the first strand 
cDNA product produced above to a 100 ul solution containing 20 mM Tris-Cl 
at pH 7.5, 100 mM KCl, 5 mM MgCl.sub.2, 10 mM (NH.sub.2).sub.2 SO.sub.4, 
10 mM DTT, 0.05 mg/ml bovine serum albumin (BSA), 50 uM of dGTP, 50 uM 
dATP, 50 uM dTTP, 50 uM dCTP, 150 uM betanicotinamide adenine dinucleotide 
(.beta.-NAD.sup.+) (Sigma Chemical Company, St. Louis, Mo.), 15 uCi/ul 
[.alpha.-.sup.32 P]dCTP, 30 units E. coli DNA polymerase, 2.5 units RNase 
H, and 4 units E. coli DNA ligase. This solution was maintained at 14C for 
1 hour and then further maintained at 25C for 1 hour. The second strand 
cDNA synthesis reaction was terminated by adding 5 ul of 0.05 M EDTA at pH 
8.0, 5 ul of 10% SDS. The nucleic acids were purified from this reaction 
mixture by phenol extraction followed by ethanol precipitation. 
The double stranded cDNA produced above was prepared for insertion into a 
cloning vector, by converting the ends of the double stranded cDNA to 
blunt ends in the following fill-in reaction. One half of the double 
stranded cDNA produced above was added to a solution containing 33.4 mM 
Tris-acetate at pH 7.8, 66.6 mM potassium acetate, 10 mM magnesium 
acetate, 0.5 mM .DTT 87.5 ug/ml BSA, 310 uM dGTP, 310 uM dATP, 310 uM 
dTTP, 310 uM dCTP and 8 units of T4 DNA polymerase. This solution was 
maintained at 37 C. for 30 minutes and the reaction terminated by adding 5 
ul of 0.05 M EDTA. The blunt-ended, cDNA produced was purified by phenol 
extraction and ethanol precipitation. 
Eco RI adaptors were annealed and then ligated to the blunt-ended cDNA 
produced above. Briefly, polynucleotide N1(Table 1) was kinased by adding 
1 ul of the polynucleotide and 20 units of T4 polynucleotide kinase to a 
solution containing 70 mM Tris-Cl at pH 7.6, 10 mM MgCl.sub.2, 5 mM DTT, 
10 mM 2Me and 500 ug/ml of BSA. The solution was maintained at 37C for 30 
minutes and the reaction stopped by maintaining the solution at 65 C. for 
10 minutes. 20 ng of polynucleotide N2(Table I was added to the above 
kinasing reaction together with 1/10 volume of a solution containing 20 mM 
Tris-Cl at pH 7.4, 2 mM MgCl.sub.2 and 15 mM NaCl. This solution was 
heated to 70 C. for 5 minutes and allowed to cool to room temperature, 
approximately 25C., over 1.5 hours in a 500 ul beaker of water. During 
this time period, the 2 polynucleotides present in the solution annealed 
to form the double stranded Eco RI adaptor. 
TABLE 1 
______________________________________ 
Eco RI Adaptor Polynucleotides 
______________________________________ 
(N1) 5'-CCTTGACCGTAAGACATG-3' 
(N2) 5'-AATTCATGTCTTACGGTCAAGG-3' 
______________________________________ 
This double stranded Eco RI adaptor was covalently linked (ligated) to the 
blunt-ended cDNA produced above by adding 5 ul of the annealed adaptors to 
a solution containing 50 ul Tris-Cl at pH 7.5, 7 ul MgCl.sub.2, 1 mM DTT, 
1 mM ATP and 10 units of T4 DNA ligase. This solution was maintained at 37 
C. for 30 minutes and then the T4 DNA ligase was inactivated by 
maintaining a solution at 72 C. for 15 minutes. 
The 5' ends of the resulting cDNA were phosphorylated by admixing 5 ul of 
the above reaction, 4 ul of a solution containing 10 mM ATP and 5 units of 
T4 polynucleotide kinase. This solution was maintained at 37 C. for 30 
minutes and then the T4 polynucleotide kinase was inactivated by 
maintaining the solution at 65 C. for 10 minutes. 
The cDNA prepared above was size fractionated to obtain long cDNA inserts 
using a method similar to the method described in Molecular Clonino: A 
Laboratory Manual, Maniatis et al., eds., Cold Spring Harbor Laboratory, 
New York (1982). Briefly, the reaction mixture prepared above was added to 
an equal volume of 2.times. CL4B column buffer consisting of 20 mM Tris-Cl 
at pH 8.0, 1.2 M NaCl, 1 mM EDTA and 0.1% sarkosyl. This solution was 
loaded onto a 5 ml CL-4B column that was previously prepared using 
pre-swollen sepharose CL-4B (Pharmacia LKB Biotechnology Inc., Piscataway, 
N.J.). The sample was allowed to enter the column and then the column was 
filled with 1.times. column buffer consisting of 10 mM Tris-Cl at pH 8.0, 
600 mM NaCl, 1 mM EDTA and 0.1% sarkosyl. The column was allowed to flow 
by gravity and approximately 200 ul fractions were collected manually. The 
size of the double stranded cDNA present in each of the fractions was 
determined by gel electrophoresis through a 0.8% agarose gel. Fractions 
containing high molecular weight cDNA as determined by the agarose gel 
electrophoreses were pooled and concentrated using butanol extraction and 
then ethanol precipitated to produce size-fractionated cDNA. 
The size-fractionated cDNA prepared above was ligated directly into lambda 
Zap (Stratagene Cloning Systems, La Jolla, Calif.) that had been 
previously digested with the restriction endonuclease Eco RI. The ligation 
mixture was packaged according to the manufacturer's instructions using 
Gigapack II gold packaging extract available from Stratagene Cloning 
Systems and plated on BB4 cells (Stratagene Cloning Systems, La Jolla, 
Calif.) to produce plaques. 
The plaques were screened with a radiolabeled probe containing the constant 
region gene of a human antibody. Briefly, the human IgG constant region 
probe previously described by Rabbitts et al., Cold Spring Harbor 
Quantitative Biology 45:867-878 (1980), and the human Kappa light chain 
probe previously described by Rabbitts et al., Cold Spring Harbor 
Quantitative Biology 45:867-878 (1980), was nick translated using standard 
protocols described by Molecular Cloning: A Laboratory Manual, Maniatis et 
al., eds., Cold Spring Harbor, N.Y. (1982). Probes prepared using this 
protocol and having a specific activity of greater than 1.times.10.sup.8 
cpm/ug were hybridized with plaques from the above-prepared library using 
methods well known to one skilled in the art. Briefly, the titer of the 
cDNA library prepared above was determined by making serial dilutions of 
the library into a buffer containing 100 mM NaCl, 50 mM Tris-Cl at pH 7.5 
and 10 mM magnesium sulfate. 10 ul of each dilution was admixed to 200 ul 
of exponentially growing E. coli cells and maintained at 37C for 15 
minutes to allow the phage to absorb to the bacterial cells. 3 ml of top 
agar consisting of 5 g/1 NaCl, 2 g/1 of magnesium sulfate, 5 g/1 of yeast 
extract, 10 g/1 of NZ Amine (casein hydrolysate) and 0.7% molten agarose 
was prepared and placed in a 50 C. water bath until used. The phage, the 
bacteria and the top agar were mixed and then evenly distributed across 
the surface of a prewarmed bacterial agar plate (5 g/1 NaCl, 2 g/1 
magnesium sulfate, 5 g/1 yeast extract, 10 g/1 NZ Amine and 15 g/1 difco 
agar. The plates were maintained at 37C for 12 to 24 hours during which 
time the lambda plaques developed on the bacterial lawn. The lambda 
plaques were counted to determine the total number of plaque forming units 
per milliliter in the original library. 
The titered cDNA library was then plated out so that replica filters could 
be produced from the library. The replica filters were used to later 
segregate the individual clones containing cDNAs coding for either 
immunoglobulin heavy or immunoglobulin light chain. Briefly, a volume of 
the titer cDNA library that would yield 20,000 plaques per 150 millimeter 
plate was added to 600 ul of exponentially growing E. coli cells and 
maintained at 37C for 15 minutes to allow the phage to absorb to the 
bacterial cells. Then 7.5 ml of top agar was added to the solution 
containing the bacterial cells and phage. The bacterial cells with the 
phage absorbed to them were mixed with the top agar and the entire mixture 
distributed evenly across the surface of the prewarmed bacterial agar 
plate. This entire process was repeated for sufficient number of plates to 
produce a total number of plaques at least equal to the library size. 
These plates were then maintained at 37 C. for 16 hours during which time 
the plaques appeared on the bacterial lawn. The plates were then overlaid 
with nitrocellulose filters and the orientation of each filter on the 
bacterial plates marked with ink dots. The filters were maintained on the 
bacterial plates for 1 to 5 minutes and then removed with a blunt-ended 
forceps and placed contact side up on a sponge pad soaked in a denaturing 
solution consisting of 1.5M NaCl and 0.5 M NaOH for approximately 1 
minute. The filter was then transferred, contact side up, onto a sponge 
pad containing a neutralizing solution consisting of 1.5 M NaCl and 0.5 M 
Tris-Cl at pH 8.0 for 5 minutes. The filter was then rinsed in a solution 
containing 0.36 M NaCl, 20 mM NaH.sub.2 PO.sub.4 at pH 7.4, and 2 mM EDTA 
and placed on Whatman 3 MM paper to dry. This process was repeated for 
each bacterial plate to produce a second replica filter for hybridization. 
After all the filters were dry the sheets were placed between Whatman 3 MM 
paper and the filter was baked for 2 hours at 80 C. in a vacuum oven. The 
filters were now ready for hybridization with specific probes. 
The baked filters were placed on the surface of a solution containing 0.9M 
NaCl and 0.09M sodium citrate at pH 7.0 until they became thoroughly 
wetted from beneath. The filters were submerged in the same solution for 5 
minutes. The filters were transferred to a pre-washing solution containing 
50 mM Tris-Cl at pH 8.0, 1M NaCl, 1 mM EDTA and 0.1% SDS. The pre-washing 
solution was then maintained at 42 C. for 2 hours. 
The filters were removed from the pre-washing solution and placed in a 
pre-hybridization solution containing 25% formamide, 1.0 M NaCl 50% 
dextran sulfate, 0.05 M NaPO. at pH 7.7, 0.005 M EDTA, 0.1% ficoll, 0.1% 
BSA, 0.1% polyvinyl pyrolidone, 0.1% SDS and 100 ug/ml denatured, salmon 
sperm DNA. The filters were maintained in the pre-hybridization solution 
for 4 to 6 hours at 42 C. with gentle mixing. The filters were then 
removed from the prehybridization solution and placed in a hybridization 
solution consisting of pre-hybridization solution containing 
2.times.10.sup.6 cpm/ml of .sup.32 P-labeled probe that had a specific 
activity of at least 1.times.10.sup.8 cpm/ug. The filters were maintained 
in the hybridization solution for 12 to 24 hours at 42 C. with gentle 
mixing. After the hybridization was complete the hybridization solution 
was discarded and the filters were washed 3 to 4 times for 10 minutes in a 
large volume of a solution containing 0.9 M NaCl, 0.09 M sodium citrate at 
pH 7.0 and 0.1% SDS at 60C. The filters were removed from the washing 
solution and air dried on a sheet of Whatman 3 MM paper at room 
temperature. The filters were taped to sheets of 3 MM paper and wrapped 
with plastic wrap and used to expose X-ray film (Kodak XR or equivalent) 
at -70C with an intensifying screen to produce an autoradiogram. The film 
was developed according to manufacturer's directions. Positive 
hybridization signals were aligned to the proper plaque by virtue of the 
asymmetrical ink spots placed on the nitrocellulose filters. 
Hybridizing plaques were isolated to purity and the inserts excised from 
the lambda ZAP vector according to the underlying in vivo excision 
protocol provided by the manufacturer, Stratagene Cloning Systems, La 
Jolla, Calif. and described in Short et al., Nucleic Acids Res., 
16:7583-7600 (1988). This in vivo excision protocol moves the cloned 
insert from the lambda ZAP vector into a phagemid vector to allow easy 
manipulation and sequencing. The hybridizing inserts were sequenced using 
the Sanger dideoxy method described by Sanger et al., Proc. Natl. Acad. 
U.S.A., 74:5463-5467 (1977) and using the Sequenase DNA Sequencing kit 
(United States Biochemical Corporation, Cleveland, Ohio). Two full length 
light chain clones designated pABZ100 and pABZ101 were identified by DNA 
sequencing. In addition, one full length heavy chain clone designated 
pABZ200 was also identified. 
These full length cDNA clones were subcloned into mp18 using procedures 
similar to the procedures described in Molecular Cloning: A Laboratory 
Manual, Maniatis et al., eds., Cold Spring Harbor Laboratory New York 
(1982). Briefly, the phagemids containing the full length cDNA clones were 
digested with the restriction endonuclease Eco RI and the full length cDNA 
inserts isolated by gel electrophoresis. The isolated full length cDNA 
inserts were ligated to M13 mp18 that had been previously digested with 
Eco RI. The ligation mixture was plated on appropriate bacterial host 
cells and phage plaques containing the full length cDNA inserts isolated. 
The accuracy of this cloning step was confirmed by restriction mapping. 
Single stranded uracil-containing template DNA was prepared according to 
the protocols provided with the Muta-Gene M13 in vitro Mutagenesis kit 
(Bio-Rad Laboratories, Richmond, Calif.). Briefly, an isolated colony of 
bacterial strain CJ236 containing both the dut and uno mutations was 
admixed into 20 ml of LB media (10 g/1 Bactotryptone, 5 g/1 yeast extract 
and 5 g/1 NaCl) containing 30 ug/ml chloramphenicol. This solution Was 
maintained at 37C for 12 to 16 hours to produce an overnight culture. 1 ml 
of this overnight culture was admixed with 50 ml of 2.times. YT medium (16 
g/l Bactotryptone, 10 g/1 yeast extract and 5 g/1 NaCl) containing 30 
ug/ml chloramphenicol in a 250 ml flask. This solution was maintained at 
37 C. with constant shaking for about 4 hours or until the optical density 
at 600 nanometers (nm) was 0.3. This optical density corresponds to 
approximately 1.times.10.sup.7 colony forming units per millimeter. The 
M13 phage containing the full length cDNA inserts were added at a 
multiplicity of infection of 0.2 or less. This solution was maintained 
with shaking at 37 C. for 4 to 6 hours. 30 ml of the resulting culture was 
transferred to a 50 ml centrifuge tube and centrifuged at 17,000 .times. g 
(12,000 revolutions per minute in the Sorvall SS-34 rotor) for 15 minutes 
at 4C. The resulting phage particle containing supernatant was transferred 
to a fresh centrifuge tube and recentrifuged at 17,000 .times. g for 15 
minutes at 4C. This second supernatant was transferred to a fresh 
polyallomer centrifuge tube and 150 micrograms of RNase A admixed to the 
supernatant. This supernatant was maintained at room temperature for 30 
minutes to allow the RNase A to digest any RNA present. One/fourth volume 
of a solution containing 3.5 M ammonium acetate and 20% polyethylene 
glycol 000 (PEG 8000) was admixed to this supernatant. This supernatant 
was maintained on ice for 30 minutes. During this time, any phage 
particles present in the supernatant were precipitated by the PEG 8000. 
The precipitated phage particles were collected by centrifuging this 
solution at 17,000 .times.g for 15 minutes at 4 C. The resulting pellet 
was resuspended in 200 ul of high salt buffer (300 mM NaCl, 100 mM Tris-Cl 
at pH 8.0 and 1 mM EDTA). This solution was maintained on ice for 30 
minutes and then centrifuged for 2 minutes in an microfuge to remove any 
insoluble material. The resulting supernatant was transferred to a fresh 
tube and stored at 4 C. until used as a phage stock. 
Single stranded uracil containing template DNA was prepared by extracting 
the entire 200 ul phage stock twice with an equal volume of neutralized 
phenol. The aqueous phase was re-extracted once with a solution of phenol 
chloroform (25:25:1 phenol: chloroform:isoamyl alcohol) and further 
extracted several times with chloroform isoamyl alcohol (1:1/48 
chloroform:isoamyl alcohol). One/tenth volume of 7.8 M ammonium acetate 
and 2.5 volumes of ethanol were admixed to the resulting aqueous phase. 
This solution was maintained at -70 C. for at least 30 minutes to 
precipitate the DNA. The precipitated DNA was collected by centrifuging 
the solution for 15 minutes at 4C. The resulting DNA pellet was washed 
once with 0% ethanol and resuspended in 20 ul of a solution containing 10 
mM Tris-Cl at pH 7.6 and 1M EDTA. The amount of uracil containing template 
DNA present in this solution was determined by gel electrophoresis. This 
uracil containing template DNA was used in further mutagenesis steps to 
introduce restriction endonuclease sites into the full length cDNAs. 
Mutagenic full length cDNAs were synthesized according to the procedures 
provided in the Muta-Gene kit (Bio-Rad Laboratories, Richmond, Calif). 
Briefly, polynucleotides designed to introduce Eco RI restriction 
endonuclease sites were used to prime the synthesis of a mutagenic strand 
from the single-stranded uracil containing template DNA. The 
polynucleotide was phosphorylated by admixing 200 picomoles (pmoles) of 
the selected polynucleotide with a solution containing 100 mM Tris-Cl at 
pH 8.0, 10 mM MgCl.sub.2, 5 mM DTT, 0.4 mM ATP and 4.5 units of T4 
polynucleotide kinase to produce a kinasing reaction. This solution was 
maintained at 37 C. for 45 minutes. The kinasing reaction was stopped by 
maintaining the solution at 65 C. for 10 minutes. The kinased 
polynucleotide was diluted to 6 moles/ul with a solution containing 10 mM 
Tris-Cl at pH 7.6 and 1 mM EDTA. 
The kinased polynucleotide was annealed to the single stranded uracil 
containing DNA template prepared above by admixing 200 ng of uracil 
containing template DNA, 3 moles of kinased polynucleotide, 20 mM Tris-Cl 
at pH 7.4, 2 mM MgCl.sub.2 and 50 mM NaCl. This solution was maintained at 
70C for 5 minutes and allowed to cool at a rate of approximately 1C per 
minute to 30C. This solution was then maintained on ice until used. 1 ul 
of a solution containing 4 mM dATP, 4 mM dCTP, 4 mM dCTP, 4 mM TTP, 7.5 mM 
ATP, 175 mM Tris-Cl at pH 7.4, 37.5 mM MgCl.sub.2, 215 mM DTT, was admixed 
to the solution along with 5 units of T4 DNA ligase and 1 unit of 4 DNA 
polymerase. This solution was maintained on ice for 5 minutes to stabilize 
the polynucleotide primer by initiation of DNA synthesis under conditions 
that favor binding of the polynucleotide to the uracil containing 
template. The solution was then maintained at 25 C. for 5 minutes and 
finally maintained at 37 C. for 90 minutes. The synthesis reaction was 
stopped by admixing 90 ul of stop buffer (10 mM Tris-Cl at pH 8.0 and 10 
mM EDTA) to this solution and freezing it. This synthesis reaction was 
then stored at -20 C. until used. 
The synthesis reaction was transformed into competent MV1190 cells using 
the protocol described in the Muta-Gene kit. Briefly, competent MV1190 
cells were prepared by admixing an isolated colony of MV1190 cells to 10 
ml of LB medium and maintaining this solution at 37C overnight with 
constant shaking. The next day, 40 ml of LB medium was admixed with a 
sufficient amount of the overnight MV1190 culture to give an initial 
absorbance reading (optical density at 00 nm) of approximately 0.1 The 
solution was then maintained at 37C for approximately 2 hours with 
constant shaking. During this time, the culture should reach an absorbance 
reading of 0.8 to 0.9. When this absorbance reading is reached, the MV1190 
cells were centrifuged at 5,000 rpm for 5 minutes at C. The MV1190 cell 
pellet was resuspended in 1 ml of ice-cold 50 mM CaCl.sub.2. An additional 
19 ml of ice-cold 50 mM CaCl.sub.2 was admixed to this solution. The 
resulting solution was maintained on ice for 30 minutes. The cells were 
centrifuged at 5,000 rpm for minutes at 0C. The MV1190 cell pellet was 
resuspended in 1 ml of ice-cold 50 mM CaCl.sub.2. An additional 3 ml of 
ice-cold 50 mM CaCl.sub.2 was admixed to the solution and the solution 
maintained on ice. The MV1190 cells were now competent for transformation. 
A 10 ul aliquot of the synthesis reaction prepared above was admixed gently 
with 0.3 ml of competent MV1190 cells in a cold 1.5 ml sterile 
polypropylene tube. This solution was maintained on ice for 90 minutes. 
The solution was then placed in a 42C water bath for 3 minutes and 
returned immediately to ice. The transformed cells were then plated on the 
MV1190 cell line at 3 different concentrations. 10 ul, 50 ul, and 100 ul 
of the transformed cells were added to individual tubes containing 0.3 ml 
of a MV1190 overnight cell culture. This solution was gently but 
thoroughly mixed and then 50 ul of 2% 
5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-GAL), 20 ul of 100 
mM isopropyl-beta-thiogalactopyranoside (IPTG) and 2.5 ml of molten top 
agar (0.7 g Bacto-Agar/100 ml in LB medium) that had been cooled to about 
50 C. was admixed to the solution. The resulting solution was immediately 
poured onto the surface of bacterial plates consisting of 15 g/L 
Bacto-Agar in LB medium. The agar was allowed to cool for about 10 minutes 
and then the plates were inverted and maintained overnight at 37 C. during 
which time plaques developed in the MV1190 cell lawn. 
Isolated plaques resulting from the above transformation were picked and 
grown up according to standard procedures described in the instruction 
provided with the Muta-Gene Kit (Bio-Rad Laboratories, Richmond, Calif.). 
Double-stranded RF DNA was then produced from each plaque using the 
alkaline lysis mini-prep procedure described in Molecular Cloning: A 
Laboratory Manual, Maniatis et al., eds., Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y. (1982). The resulting DNA was then digested with 
restriction endonucleases that allow the identification of mutants 
containing the desired polynucleotide. 
Mutants identified in this manner were then sequenced to confirm the DNA 
sequence of the mutant cDNA coding for either immunoglobulin heavy chain 
or immunoglobulin light chain. 
2. Construction Of Expression Vectors Containing Kappa Light Chain Genes 
An expression vector containing the entire kappa light chain gene including 
the kappa leader was produced in the following manner. The full length 
kappa light gene cDNA isolated above was mutagenized using polynucleotides 
P1 and P3 (Table 2) and the mutagenesis procedures described above. 
Polynucleotide P1 introduces an Eco RI restriction endonuclease site at 
the 5' end of the full length kappa cDNA. Polynucleotide P3 introduces an 
Eco RI restriction endonuclease site at the 3' end of the full length 
kappa light chain cDNA clone. Mutant transformants containing 2 additional 
Eco RI restriction endonuclease sites indicating that both polynucleotide 
P1 and polynucleotide P3 had been introduced into the mutants were 
isolated. These mutants were then sequenced to confirm that they did 
contain the DNA sequence of both polynucleotide P1 and polynucleotide P3. 
The full length kappa light chain cDNA (FIG. 1A) was excised with the 
restriction endonuclease Eco RI sites at the 5' and 3' ends and the 
restriction fragment isolated using gel electrophoresis. This isolated 
restriction fragment was directly ligated to the pMON530 expression vector 
that had been previously digested with Eco RI (FIG. 2). The resulting 
ligation mixture was transformed into suitable host cells and individual 
transformants isolated. DNA was prepared from the individual transformants 
using procedures similar to the standard of procedures described in 
Molecular Cloning: A Laboratory Manual, Maniatis et al., Cold Spring 
Harbor Laboratory, New York (1982). The transformant DNA was then digested 
with various restriction endonucleases to establish the orientation of the 
kappa light chain cDNA gene within the expression vector. The resulting 
kappa light chain expression vector contained a gene coding for the entire 
kappa chain including the kappa leader. 
An expression vector containing the kappa light chain gene without its 
leader sequence was produced in the following manner. The full length 
kappa light chain genes cDNA isolated above was mutagenized using 
polynucleotides P2 and P3 (Table 2) and the mutagenesis described above. 
Polynucleotide P2 introduces an Eco RI restriction endonuclease site just 
5' of the sequence that codes for the N-terminal amino acid of the mature 
kappa light chain and thus removes the kappa light chain leader sequence 
normally transcribed in the wild type cDNA. Polynucleotide P3 introduces 
an Eco RI restriction endonuclease site at the 3' end or the full length 
kappa light chain cDNA clone. Mutant transformants containing 2 additional 
Eco RI restriction endonuclease sites indicating that both polynucleotide 
P2 and polynucleotide P3 had been introduced into the mutants were 
isolated. These mutants were then sequenced to confirm that they did, in 
fact, contain the DNA sequence of both polynucleotide P2 and 
polynucleotide P3. 
The leaderless kappa light chain cDNA produced by this mutagenesis was 
excised with the restriction endonuclease Eco RI sites at the 5' and 3' 
ends and the restriction fragment isolated using gel electrophoresis. This 
isolated restriction fragment was directly ligated to the pMON530 
expression vector that had been previously digested with Eco RI (FIG. 2). 
The resulting ligation mixture was transformed into suitable host cells 
and individual transformants isolated. DNA was prepared from the 
individual transformants and the transformant DNA was then digested with 
various restriction endonucleases to establish the orientation of the 
leaderless kappa light chain cDNA gene within the expression vector. The 
resulting leaderless kappa light chain expression vector contained a gene 
coding for the kappa chain without its normal leader sequence. 
TABLE 2 
__________________________________________________________________________ 
Mutagenic Polynucleotides 
__________________________________________________________________________ 
(P1)-5'-TGTGAAAACCATATTGAATTCCACCAATACAAA-3' 
(P2)-5'-ATTTAGCACAACATCCATGTCGACGAATTCAATCCAAAAAAGCAT-3' 
(P3)-5'-GGGGAGCTGGTGGTGGAATTCGTCGACCTTTGTCTCTAACAC-3' 
(P4)-5'-CCATCCCATGGTTGAATTCAGTGTCGTCAG-3' 
(P5)-5'-CTGCAACTGGACCTGCATGTCGACGAATTCAGCTCCTGACAGGAG-3' 
(P6)-5'-CCTGTAGGACCAGAGGAATTCGTCGACACTGGGATTATTTAC-3' 
__________________________________________________________________________ 
3. Construction Of Expression Vectors Containing Gamma Heavy Chain Gene 
An expression vector containing the entire gamma heavy chain gene including 
the gamma leader was produced in the following manner. The full length 
gamma heavy chain gene cDNA isolated above was mutagenized using 
polynucleotides P4 and P6 (Table 2) and the mutagenesis procedures 
described above. Polynucleotide P4 introduces an Eco RI restriction 
endonuclease site at the 5' end of the native full length gamma cDNA. 
Polynucleotide P6 introduces an Eco RI restriction endonuclease site at 
the 3' end of the full length gamma heavy chain cDNA clone. Mutant 
transformants containing 2 additional Eco RI restriction endonuclease 
sites indicating that both polynucleotide P4 and polynucleotide P6 had 
been introduced into the mutants were isolated. These mutants were then 
sequenced to confirm that they did in fact contain the DNA sequence of 
both polynucleotide P4 and polynucleotide P6. 
The full length gamma heavy chain cDNA was excised with the restriction 
endonuclease Eco RI at the 5' and 3' ends (FIG. 1B) and the restriction 
fragment isolated using gel electrophoresis. This isolated restriction 
fragment was directly ligated to the pMON530 expression vector that had 
been previously digested with Eco RI (FIG. 2). The resulting ligation 
mixture was transformed into suitable host cells and individual 
transformants isolated. DNA was prepared from the individual transformants 
and the transformant DNA was then digested with various restriction 
endonucleases to establish the orientation of the gamma heavy chain cDNA 
within the expression vector. The resulting gamma heavy chain expression 
vector contained a gene coding for the entire gamma heavy chain including 
the gamma leader. 
An expression vector containing the gamma heavy chain gene without its 
leader sequence was produced in the following manner. The full length 
gamma heavy chain gene cDNA isolated above was mutagenized using 
polynucleotides P5 and P6 (Table 2) and the mutagenesis procedures 
described above. Polynucleotide P5 introduces an Eco RI restriction 
endonuclease site immediately 5' of the sequences that code for the 
N-terminal amino acid of the mature protein and thus remove the normal 
gamma leader sequence. Polynucleotide P6 introduces and Eco RI restriction 
endonuclease site at the 3' end of the full length gamma heavy chain cDNA 
clone. Mutant transformants containing 2 additional Eco RI restriction 
endonuclease sites indicating that both polynucleotide P5 and 
polynucleotide P6 had been introduced into the mutants were isolated. 
These mutants were then sequenced to confirm that they did contain both 
polynucleotide P5 and polynucleotide P6. 
This leaderless gamma heavy chain cDNA was excised with the restriction 
endonuclease Eco R1 sites located at the 5' and 3' ends and the resulting 
restriction fragment isolated using gel electrophoresis. This isolated 
restriction fragment was directly ligated to the pMON530 expression vector 
that had been previously digested with Eco RI (FIG. 2). The resulting 
ligation mixture was transformed into suitable host cells and individual 
transformants isolated. DNA was prepared from the individual transformants 
and the transformant DNA was then digested with various restriction 
endonucleases to establish the orientation of the gamma heavy chain cDNA 
within the expression vector. The resulting gamma heavy chain expression 
vector contained a gene coding for the gamma heavy chain without its 
native gamma leader. 
4 Introduction Of Immunoglobulin Genes Into Plants 
The leaderless kappa expression vector, the leaderless gamma expression 
vector, the native kappa expression vector and the native gamma expression 
vector prepared in the above examples were mobilized into Agrobacterium 
strain GV3111-SE using the triparental conjugation system of Ditta et al., 
Proc. Natl. Acad. Sci. USA, 77:7347-7351 (1980). Briefly, the 
Agrobacterium (acceptor) GV3111-SE, was grown on an agar plate containing 
MGL medium consisting of 2.6 g/L yeast extract, 5 g/L tryptone, 5 g/L 
NaCl, 5 g/L mannitol, 1.16 g/L monosodium glutamate, 0.25 g/L KH.sub.2 
PO.sub.4, 0.1 g/L MgSO.sub.4 -7H.sub.2 O per liter, and 1 mg/L biotin at 
pH 7.0 for 12 to 18 hr at 28 C. The E. coli (helper) strain containing the 
mobilization plasmid pRK2073 described by Better et al., J. Bacteriol, 
155:311 (1983), was grown on an agar plate containing LB agar (LB agar is 
5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 15 g/L Bacto-agar, at 
pH 7.0) for 12 to 18 hr at 37 C. The E. coli containing each of the 
expression vectors were grown on bacterial culture plates containing LB 
medium supplemented with 3 ug/ml tetracycline and 10 ug/ml kanamycin for 
12 to 18 hr at 37 C. An equal amount (about 1.times.10.sup.8 cells) of all 
three bacteria, the acceptor Agrobacterium, the helper E. coli, and the E. 
coli containing the expression vectors were mixed together and plated out 
on a bacterial plate containing AB agar medium containing 100 ug/ml 
kanamycin, 200 ug/ml spectinomycin and 50 ug/ml chloramphenicol (1 liter 
AB medium agar contains 1 g NH.sub.4 Cl, 0.3 g MgSO.sub.4 -7H.sub.2 O, 
0.15 g KCl, 0.01 g CaCl.sub.2, 2.5 mg FeS.sub.4 -7H.sub.2 O, 3 g K.sub.2 
HPO.sub.4, 1.15 g NaH.sub.2 PO.sub.4 -H.sub.2 O, 5 g glucose and 15 g 
Bacto-agar). The bacterial culture plates were incubated at 28 C. for two 
to four days. Single transformant colonies were admixed into a culture 
flask containing LB medium supplemented with 100 ug/ml kanamycin, 200 
ug/ml spectinomycin and 50 ug/ml, chloramphenicol, which was then 
maintained with gentle shaking at 28C for 12 to 18 hours. Each of the 
expression vectors prepared in the above examples were now in a culture of 
Aorobacterium and thus ready to be introduced into a plant. 
Tobacco leaf discs were transformed using the methods described in Rogers 
et al., in Methods For Plant Molecular Biology, Academic Press, Inc., San 
Diego (1988). Healthy, young tobacco leaves were surface sterilized by 
placing the leaves in a solution containing 20% household bleach (w/v) and 
0.1% SDS (w/v) for 8 minutes. The leaves were then transferred to a 
solution containing 98% ethanol for 60 seconds and rinsed twice in sterile 
double distilled H.sub.2 O. The leaf discs were then punched with a 6-mm 
paper punch. The discs were placed basal side down, in MS10 solution (MS 
salts, Gibco Laboratories, Grand Island N.Y., 0.01 mg/ml thiamine HCL, 
0.001 mg/ml pyridoxine HCl, 0.001 mg/ml nicotinic acid, and 0.1 mg/ml 
inositol, 30 g sucrose, 0.01 ug/ml naphthalene acidic acid [NAA], 1.0 
ug/ml benzyladenine [BA], and 10 g/1 Bacto-agar at pH 6.0). Each disc was 
admixed to the culture of Aqrobacterium containing the expression vectors 
for 5 seconds. The discs were then blotted dry on sterile filter paper and 
transferred basal side down to the MS10 medium and the medium maintained 
for 48 hours under normal growing conditions. Each leaf disc was then 
washed in sterile water to remove most of the Aorobacterium containing the 
expression vector. The leaf discs were blotted dry on sterile number 9 
Whatman filter paper and then placed basal side up on MS10 medium 
selection plates containing 200 ug/ml kanamycin sulfate and 500 ug/ml 
carbenicillin. Selection plates were maintained under normal growing 
conditions for two weeks. Within the two weeks, callus appeared and 
shortly later shoots appeared. After the shoots appeared, they were 
transferred to regeneration plates containing MS0 medium (MSIO with no NHA 
or BA) and 200 ug/ml kanamycin sulfate and 500 ug/ml carbenicillin. The 
shoots that rooted in the regeneration plates were transferred to soil to 
produce plantlets. The plantlets were maintained under standard growth 
conditions until they reached maturity. 
A population of plantlets was prepared from each of the expression vectors 
constructed in the above examples using the procedure just outlined. Leaf 
extracts from each of the plantlet populations were screened for the 
presence of immunoglobulin heavy or light chain using an ELISA assay based 
on the methods described by Engvall et al., J. Immunol., 109:129-135 
(1972). Briefly, 50 ul of a solution containing 150 mM NaCl and 20 mM 
Tris-Cl at pH 8.0 (TBS), and either a goat anti-mouse heavy chain or a 
goat anti-mouse light chain specific IgG (Fisher Scientific, Pittsburgh, 
PA) was admixed into the wells of microtiter plates. The plates were 
maintained for about 16 hours at 4C to permit the goat antibodies to 
adhere to the microtiter well walls. After washing the wells four times 
with H.sub.2 O, 200 ul of TBS containing 5% non-fat dry milk was admixed 
to the microtiter wells. The wells were maintained for at least 30 minutes 
at 20C, and then the wells were emptied by shaking and blotted dry to form 
a solid support, i.e., a solid matrix to which the goat antibodies were 
operatively attached. 
Leaves from each of the transformants were homogenized in a mortar and 
pestle after removing the midvein. One-fourth volume of 5.times. TBS (750 
mM NaCl and 100 mM Tris-Cl at pH 8.0) was admixed to the homogenized 
transformant leaves. Two-fold serial dilutions of the homogenate were made 
in TBS (150 mM NaCl and 20 mM Tris-Cl at pH 8.0). 50 ul of the two-fold 
serial dilutions were added to each separate microtiter well and the wells 
maintained for 18 hours at 4C to permit the formation of solid-phase 
immunoreaction products. The wells were then washed with room temperature 
distilled water. 50 ul of a 1:1000 dilution of either goat anti-mouse 
heavy chain or goat anti-mouse light chain specific antibody conjugated to 
horse radish peroxidase (HRPO) (Fisher Scientific, Pittsburgh, Pa.) in TBS 
was admixed to each of the microtiter wells. The wells were maintained for 
2 hours at 37C followed by detection according to the manufacturer's 
instructions. Control microtiter wells were produced in a similar fashion 
and contained extracts from plants transformed with the vector alone and 
did not express any detectable immunoglobulin products. 
The immunoglobulin content of each plantlet was determined at least twice 
and the values shown in Table 3 are given as mean values. At least 9 
plantlets from each population of plantlets were assayed in this manner. 
The plantlets expressing either immunoglobulin heavy chain or 
immunoglobulin light chain were now shown to be transformed with the 
immunoglobulin genes and are thus termed transformants or transgenic 
plants. 
TABLE 3 
______________________________________ 
Expression of Immunoglobulin Gamma and 
Kappa Chains in Tobacco 
______________________________________ 
Gamma-NL Gamma-L 
30 .+-. 16 1412 .+-. 270 
(60) (2400) 
Kappa-NL Kappa-L 
1.4 .+-. 1.2 56 .+-. 5 
(3.5) (80) 
______________________________________ 
The results present in Table 3 demonstrate the importance of a signal 
sequence for the accumulation of the individual immunoglobulin chains. 
Kappa chain accumulation was 40-fold greater (on average) when the signal 
sequence was present in the cDNA construct; Gamma chain accumulation was 
47-fold greater. 
5 Producing a Population of Progeny Expressing Both Immunoglobulin Heavy 
and Immunoglobulin Light Chain 
Transformants produced according to Example 4 expressing individual 
immunoglobulin chains were sexually crossed to produce progeny expressing 
both chains. Briefly, the hybrid progeny were produced by was to 
emasculating immature flowers by removing the anthers from one 
transformant expressing one immunoglobulin chain to produce a female 
transformant. The female transformant was then cross-pollinated from the 
other transformant (male) expressing the other immunoglobulin chain. After 
cross-pollination, the female transformant was maintained under normal 
growing conditions until hybrid seeds were produced. The hybrid seeds were 
then germinated and grown to produce hybrid progeny containing both the 
immunoglobulin heavy chain and the immunoglobulin light chain. A schematic 
diagram of producing hybrid progeny is shown in FIG. 3. The leaves were 
homogenized and the homogenate assayed for immunoglobulin heavy chain or 
light chain expression using the ELISA assay described in Example 4. The 
number of hybrid progeny expressing immunoglobulin heavy chain 
immunoglobulin light chain, both chains or no chains (null) is shown in 
Table 4. The hybrid progeny produced from the cross of the transformants 
expressing the Kappa leader construct and the gamma leader construct 
contained assembled immunoglobulin molecules containing both gamma heavy 
chains and kappa light chains. 
TABLE 4 
______________________________________ 
Expression of Immunoglobulin Gamma and 
Kappa Chains in Hybrid Progeny 
______________________________________ 
Gamma-L Gamma-NL 
(Kappa-L) (Kappa-NL) 
3330 .+-. 2000 32 .+-. 26 
(12800) (60) 
Kappa-L Kappa-NL 
(Gamma-1) (Gamma-NL) 
3700 .+-. 2300 6.5 .+-. 5 
(12800) (20) 
______________________________________ 
TABLE 5 
______________________________________ 
Expression and Assembly of Immunoglobulin 
Gamma and Kappa Chains in Hybrid Progeny 
Gamma Kappa Gamma 
only only Kappa null 
______________________________________ 
Kappa-NL .times. 
4 6 3 5 
Gamma-NL (0% assembly) 
Kappa-L .times. 
3 10 11 4 
Gamma-L (95 .+-. 16% assembly) 
______________________________________ 
The results presented in Tables 4 & 5 demonstrate the importance of 
assembly of the two immunoglobulin chains. Compared to the parental 
transformants, the progeny that express both immunoglobulin chains 
together accumulate for more of each chain. On average, gamma chain showed 
a 2.5-fold increase in accumulation and kappa chain a 66-fold increase. 
Compared to the transformants expressing cDNAs without leader sequences, 
the increased accumulation as a result of both the leader sequence and 
dual expression resulting from the sexual cross was surprisingly large. 
Gamma chains increased by 110-fold and kappa chains by 2,600-fold. 
6. Detection of Immunoglobulin Heavy Chain-coding Genes and Immunoglobulin 
Light Chain-Coding Genes in the Transgenic Plants 
The presence of immunoglobulin heavy chain-coding genes or immunoglobulin 
light chain-coding chains in the transgenic plants and hybrid progeny was 
demonstrated by analyzing DNA extracted from the transgenic plants using 
the Southern blot procedure described in Maniatis et al., Molecular 
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory (1982). 
Briefly, DNA was extracted from 1 g of mature leaf tissue harvested from 
either the heavy chain gene transformants, the light chain gene 
transformants or the hybrid progeny after freezing the leaf segments in 
liquid nitrogen. The frozen leaf segments were homogenized in urea mix 
(420 g/L urea, 312.5 mM NaCl 50 mM Tris-Cl at pH 8.0, 20 mM EDTA and 1% 
sarcosine) with a mortar and pestle according to the procedures described 
by Shure, et al., Cell. 25:225-233 (1986). The leaf homogenate was 
extracted with phenol:CHCl.sub.3 (1:1 v/v) and the nucleic acids were 
precipitated by adding 1/6 volume of 4.4M ammonium acetate at pH 5.2 and 
one volume of isopropyl alcohol and then maintaining the resulting 
solution at -20 C. for 30 minutes. The solution containing the 
precipitated nucleic acid was centrifuged for 15 minutes at 7500 .times. g 
at 4C to collect the precipitated nucleic acid. The nucleic acid pellet 
was resuspended in a TE solution containing 10 mM Tris-Cl at pH 7.6 and 1 
mM EDTA. The concentration of DNA in the resulting solution was determined 
by spectrophotometry. 
DNA was prepared from each of the transformants using the above methods and 
20 ug of transformant DNA was digested with the restriction endonuclease 
Hind III under conditions recommended by the manufacturer, Stratagene 
Cloning Systems, La Jolla, Calif. The resulting restriction endonuclease 
fragments were size fractionated on an agarose gel and blotted to 
nitrocellulose using the methods described in Maniatis et al., Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982). 
Briefly, after the DNA had been size fractionated by electrophoresis 
through an agarose gel, the DNA was stained with ethidium bromide and a 
photograph of the gel produced. The gel containing the DNA was placed in a 
solution containing 1.5M NaCl and 0.5 M NaOH for one hour at room 
temperature with constant gentle stirring. The gel was then placed in a 
solution containing 1M Tris-Cl at pH 8.0 and 1.5M NaCl for one hour at 
room temperature with constant gentle stirring. The pH of the gel was 
periodically checked by removing a small piece of the gel and determining 
its pH in a small volume of distilled H.sub.2 O. When the gel had reached 
a pH of approximately 8.0 the gel was placed upon a thick wick soaked with 
a solution containing 87.65 g/L NaCl, 13.8 g/L NaH.sub.2 PO.sub.4 -H.sub.2 
O and 3.7 g/L EDTA at pH 7.4 (10.times. SSC). A piece of nitrocellulose 
filter (Schleicher and Schuell BA 85, Keene, NH) that had been previously 
cut to the same size as the gel and soaked in a solution containing 
10.times. SSC was placed upon the gel and any intervening air bubbles 
removed. Two pieces of Whatman 3 MM paper, cut to exactly the same size as 
the nitrocellulose filter were soaked in 2.times. SSC (2.times. SSC 
contains 17.53 g/L NaCl, 2.76 g/L NaH.sub.2 PO.sub.4 -H.sub.2 O and 0.74 
g/L EDTA at pH 7.4) and placed on top of the nitrocellulose filter and any 
intervening air bubbles removed. A stack of paper towels (5-8 centimeters 
high) cut to a size just slightly smaller than the Whatman 3 MM paper was 
placed on top of the Whatman 3 MM paper. A glass plate was placed on top 
of the resulting stack and a 500 gram weight placed on top of the plate. 
The resulting capillary action was allowed to proceed for 12 to 24 hours 
and this action transferred the DNA from the gel onto the nitrocellulose 
filter. The stack was disassembled and the nitrocellulose filter soaked in 
6.times. SSC (6.times. SSC is 52.59 g/L NaCl, 8.28 g/L NaH.sub.2 PO.sub.4 
-H.sub.2 O and 2.22 g/L EDTA at pH 7.4) at room temperature for five 
minutes. The filter was placed upon a piece of dry Whatman 3 MM paper and 
allowed to air dry. The dried filter was placed between two sheets of 3 MM 
paper and baked for 2 hours at 80 C. under vacuum to operatively link the 
DNA to the nitrocellulose filter. 
The baked filters were placed on the surface of a solution containing 0.9M 
NaCl and 0.09M sodium citrate at pH 7.0 until they were thoroughly wetted 
from beneath. The filters were submerged in the same solution for 5 
minutes. 
The filters were placed in a pre-hybridization solution containing 50% 
formamide, 0.9 M NaCl, 0.05 M NaPO.sub.4 at pH 7.7, 0.005 M EDTA, 0.1% 
ficoll, 0.1% BSA, 0.1% poly(vinyl pyrrolidone), 0.1% SDS and 100 ug/ml 
denatured, salmon sperm DNA. The filters were maintained in the 
prehybridization solution for 12 to 18 hours at 42 C. with gentle mixing. 
The filters were then removed from the pre-hybridization solution and 
placed in a hybridization solution consisting of pre-hybridization 
solution containing 1.times.10.sup.6 cpm/ml of .sup.32 P-labeled gamma 
chain probe (the entire gamma expression vector was labeled) and 
1.times.10.sup.6 cpm/ml of .sup.32 P-labeled kappa chain probe (the 
entire expression kappa vector was labeled. The filters were maintained in 
the hybridization solution for 12 to 24 hours at 42C with gentle mixing. 
After the hybridization was complete the hybridization solution was 
discarded and the filters washed 4 times for 10 minutes per wash in a 
large volume of a solution containing 0.3 M NaCl, 0.03M sodium citrate at 
pH 7.0 and 0.1% SDS at room temperature. The filters were then washed 
twice for 1.5 hours in a solution containing 0.15 M NaCl, 0.05M sodium 
citrate at pH 7.0, and 0.1% SDS at 65 C. The filters were further washed 
by transferring them to a solution containing 0.2.times. SSC (0.03M NaCl 
and 0.003M sodium citrate at pH 7.0) and 0.1% SDS at 42C for 1 hour with 
gentle agitation. The filters were removed from the washing solution and 
air dried on a sheet of Whatman 3 MM paper at room temperature. The 
filters were then taped to sheets of 3 MM paper and wrapped with plastic 
wrap and used to expose X-ray film (Kodak XR or equivalent) at -70 C. with 
an intensifying screen to produce an autoradiogram. The film was developed 
according to manufacturer's directions. 
The resulting autoradiogram is shown in FIG. 4A. The hybridizing DNA 
fragments detected in the transformants expressing either the kappa light 
chain cDNA without a leader sequence (Lane 1) or with its native leader 
sequence (Lane 3) are shown. The hybridizing DNA fragment detected in DNA 
from transformants expressing either the gamma heavy chain cDNA without a 
leader sequence (Lane 2) or with its native leader sequence (Lane 4) are 
shown. The hybridizing DNA fragments detected in hybrid progeny containing 
both kappa light chain with its native leader and gamma heavy chain with 
its leader (Lane 5) are shown. 
7. Detection Of mRNA Coding For Immunoglobulin Heavy And Light Chains In 
The Transgenic Plants 
The presence of mRNA coding for immunoglobulin heavy chain or 
immunoglobulin light chain gene in the transgenic plants and hybrid 
progeny was demonstrated by analyzing RNA extracted from the transgenic 
plants using procedures similar to those described by Molecular Cloning, A 
Laboratory Manual, supra. Briefly, RNA was extracted from 1 g of mature 
leaf tissue harvested from either the heavy chain gene transformants, the 
light chain gene transformants or the hybrid progeny. The leaf tissue was 
cut into small pieces and admixed to 10 ml of a solution containing 10 ml 
of 0.1 M Tris-Cl at pH 9.0 and phenol saturated with this buffer. The leaf 
tissue was immediately homogenized in the solution using a Polytron 
homogenizer at high speed for 1 minute. The homogenate was centrifuged at 
4,000 .times. g for 15 minutes at room temperature. The resulting aqueous 
phase was recovered and the RNA precipitated by admixing 1 ml of 3M sodium 
acetate at pH 5.2 and 25 ml of isopropanol. This solution was maintained 
at -20 C. for 20 minutes to precipitate the centrifuging this solution at 
4,000 .times. g for 15 minutes at 4C. The resulting RNA pellet was 
resuspended in 400 ul of DEPC-H.sub.2 O and transferred to a 1.5 ml 
Eppendorf tube. This solution was centrifuged in an Eppendorf microfuge 
for 5 minutes at top speed. The resulting supernatant was transferred to a 
new eppendorf tube and 40 ul of 3M sodium acetate at pH 5.2 and 1 ml of 
absolute ethanol admixed to it. This solution was maintained at -20 C. for 
20 minutes and then centrifuged for 5 minutes in an eppendorf microfuge. 
The resulting RNA pellet was resuspended in 400 ul of DEPC-H.sub.2 O and a 
small aliquot removed to determine the RNA concentration by absorbance at 
260 nm. The remainder of the solution was frozen at -70 C. until used. 
The RNA prepared above was size fractionated on denaturing formaldehyde 
agarose gels and transferred to nylon membrane. The procedures used were 
similar to the procedures described in Molecular Cloning: A Laboratory 
Manual, Maniatis et al., eds., Cold Spring Harbor Laboratories, Cold 
Spring Harbor, N.Y. (1982). Briefly, the denaturing formaldehyde agarose 
gel was prepared by melting 1.4 g of agarose in 73.3 ml of DEPC--H.sub.2 O 
water and cooling this solution to 60C in a water bath. 10 ml of a buffer 
containing 50 mM NaH.sub.2 PO.sub.4, 50 mM Na.sub.2 HPO.sub.4, 50 mM 
sodium acetate and 10 mM EDTA was admixed to this solution. 16.66 ml of 
37% formaldehyde was also admixed to the solution and the solution poured 
into a gel mold and allowed to solidify. The denaturing formaldehyde 
agarose gel was now ready for use. 
A 20 ug aliquot of RNA prepared above was admixed to 15 ul of formamide, 5 
ul of 37% formaldehyde and 3 ul of a buffer containing 50 mM NaH.sub.2 
PO.sub.4, 50 mM Na.sub.2 HPO.sub.4, 50 mM sodium acetate and 10 mM EDTA. 
This solution was maintained at 55C for 15 minutes and then immediately 
placed on ice. One/tenth volume of a solution containing 50% glycerol 1 mM 
EDTA 0.4% bromophenol blue and 0.4% xylene cyanol was thoroughly admixed 
to this solution and the solution loaded onto the denaturing formaldehyde 
gel prepared above. The gel was electrophoresed in a buffer containing 5 
mM NaH.sub.2 PO.sub.4, 5 mM Na.sub.2 HPO.sub.4, 5 mM sodium acetate and 1 
mM EDTA for 2 hours at room temperature. After the electrophoresis was 
complete the gel was soaked in several changes of water for 10 to 15 
minutes. The gel was then placed in a solution containing 0.1M Tris-Cl at 
pH 7.5 for 45 minutes. The gel was then placed in a solution containing 3M 
NaCl and 0.3M sodium citrate at pH 7.0. The gel was then placed on a thick 
wick soaked with a solution containing 1.5M NaCl and 0.15M sodium citrate 
at pH 7.0. A sheet of nylon membrane (Hybond-N, Amersham Corporation, 
Arlington Heights, Ill.) that had been previously cut to the same size as 
the gel and soaked in a solution containing 10.times. SSC was placed on 
the gel and any intervening air bubbles removed. Two pieces of Whatman MM 
paper, cut to exactly the same size as the nylon membrane were soaked in 
2.times. SSC (0.3M NaCl and 0.03M sodium citrate at pH 7.0) and placed on 
top of the nylon membrane and any intervening air bubbles removed. A stack 
of paper towels (5-8 cm high) cut to a size just slightly larger than the 
Whatman 3 MM paper was placed on the top of the Whatman 3 MM paper. A 
glass plate was placed on top of the resulting stack and a 500 g weight 
placed on top of the plate. The resulting capillary action was allowed to 
proceed for 12 to 24 hours and this action transferred the RNA from the 
gel to the nylon membrane. The stack was disassembled and the nylon 
membrane soaked in 6.times. SSC (0.9 M NaCl and 0.09 M sodium citrate at 
pH 7.0) at room temperature for 5 minutes. The nylon membrane was then 
placed on a ultraviolet radiation box for 10 minutes to operatively link 
the RNA to the nylon membrane. 
RNA containing either kappa light chain coding sequences or gamma heavy 
chain coding sequences was detected by prehybridizing and hybridizing the 
nylon membrane using the protocol described in Example 6. The resulting 
autoradiogram is shown in FIG. 4B. The hybridizing RNA species detected in 
RNA from transformants expressed either the kappa light chain cDNA without 
a leader sequence (Lane 1) or with its native leader sequence (Lane 3) are 
shown. The hybridizing RNA species detected in RNA from transformants 
expressing either the gamma heavy chain cDNA without a leader sequence 
(Lane 2) or its native leader sequence (Lane 4) are shown. The hybridizing 
RNA species detected in hybrid progeny containing both kappa light chain 
with its native leader and gamma heavy chain with its native leader (Lane 
5) are shown. 
8. Detection of Immunoglobulin Chains In The Transgenic Plants 
The expression of immunoglobulin heavy and light chains in the transgenic 
plants and hybrid progeny was demonstrated by Western blotting in which 
both heavy and light chains were detected. Using the Western blot 
procedure described in Antibodies: A Laboratory Manual, Harlow & Lane, 
eds., Cold Spring Harbor Laboratories, New York (1988). Briefly, 1 g of 
leaf segments from mature plants were homogenized in a mortar and pestle 
with 1 ml of 0.05 M Tris-Cl at pH 7.5, and 1 mM phenylmethylsulfonyl 
fluoride (PMSF) ul of the resulting leaf extract was admixed to a solution 
with a final concentration of 4M urea and 1% SDS with or without 2 mM DTT 
as indicated and the solution boiled for 3 minutes. After boiling this 
solution was electrophoresed through a 10% polyacrylamide gel containing 
SDS (SDS-PAGE) as described in NH. Chua, Methods in Enzymol. 69:434-446 
(1980). The electrophoresed proteins were then transferred (affixed) to a 
sheet of nitrocellulose as described in Antibodies: A Laboratory Manual, 
Harlow and Lane, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y. (1988). Briefly, the nitrocellulose sheet was placed in a solution 
containing 20 mM Tris-Cl at pH 8.0, 150 mM NaCl and 0.01% polyoxyethylene 
sorbitan monolaurate (Tween 20) (TBST) containing 5% bovine serum albumin 
(BSA) and 0.5% non-fat dried milk. The nitrocellulose sheet was maintained 
in this solution for 6 hours at 4 C. The nitrocellulose was then placed in 
a solution containing a 1:500 dilution of a biotinylated goat anti-mouse 
whole IgG antibody (Cappel, Malvery, Pa.) in TBST and the solution 
containing the nitrocellulose sheet maintained at 4 C. for 24 hours. 
During this time, the immunoglobulin heavy chains and the immunoglobulin 
light chains immobilized on the nitrocellulose sheet immunoreacted with 
the biotinylated goat anti-mouse whole IgG antibody to form an 
immunoreaction product on the nitrocellulose sheet. The nitrocellulose 
sheet was removed from this solution and washed with TBST solution and 
then placed in a TBST solution containing streptavidin-conjugated alkaline 
phosphatase (Fisher Scientific, Pittsburgh, Pa.). This solution was 
maintained for 1 hour at 25 C. The nitrocellulose sheet was removed from 
this solution and washed with TBST. 
The immunoreaction product was visualized by maintaining the nitrocellulose 
sheet in a solution containing 100 mM Tris-Cl at pH 9.5, 100 mM NaCl, 5 mM 
MgCl.sub.2, 0.3 mg/ml of nitro blue tetrazolium (NBT) and 150 ug/ml 
5-bromyl-4-chloryl-3-indolyl phosphate (BCIP) for 30 minutes at room 
temperature. The residual color development solution was rinsed from the 
filter with a solution containing 20 mM Tris-Cl at pH 7.5 and 150 mM NaCl. 
The filter was then placed in a stop solution consisting 1 mM EDTA, pH8. 
The development of an intense purple color indicated the location of the 
immunoreaction products. 
Expression of immunoglobulin heavy chain in the heavy chain transformants, 
immunoglobulin light chain in the light chain transformants and both 
immunoglobulin heavy and light chains in the hybrid progeny was 
demonstrated using the Western blot (FIG. 5). In addition, the 
immunoglobulin heavy and light chains produced in the hybrid progeny were 
assembled into immunoglobulin molecules as evidenced by the high molecular 
weight immunoreactive gamma and kappa chain seen under non-reducing 
conditions (FIG. 5). 
9. Immunoglobulin Molecules Expressed In The Transgenic Plants Bind Antigen 
The binding of antigen by the immunoglobulin molecules expressed in the 
transgenic plants was demonstrated using an ELISA assay similar to the 
ELISA assay described in Example 4. This antigen binding ELISA assay was 
modified in the following manner. Instead of adhering the goat antibodies 
to the microtitre well walls, the antigen P3 conjugated to BSA according 
to the methods described in Tramontano et al., Proc. Natl. Acad. Sci. USA, 
83:6736-6740 (1986), was adhered to the microtitre well walls. Leaf 
homogenate from each of the plantlet populations were then added to the 
wells and the binding of the immunoglobulin molecules present in the 
homogenate detected using goat anti-mouse heavy chain conjugated to HRPO 
as described in Example 4. 
The immunoglobulin molecules expressed in the transgenic plant directly 
bound their specific antigen, P3 in this antigen binding ELISA assay. To 
demonstrate the specificity of this antibody antigen interaction, an 
additional competitive ELISA assay was performed. This assay was similar 
to the antigen binding ELISA assay described above except that before the 
serial dilutions of leaf homogenate, 5 ul of a 500 uM solution of P3 was 
added to a duplicate well to act as a competitor for antibody binding to 
P3-BSA adhered to the microtitre well walls. The remainder of this 
competition ELISA assay was carried out according to Example 4. 
The interaction between the antibodies expressed in the transgenic plants 
and their specific antigen, P3, was specifically inhibited by free antigen 
in this competition ELISA assay. 
10. Catalytic Activity Immunoglobulin Expressed in Transgenic Plants 
The catalytic activity of immunoglobulin molecules expressed in transgenic 
plants was demonstrated by purifying the 6D4 immunoglobulin molecule from 
tobacco plants expressing the functional immunoglobulin and assaying the 
purified immunoglobulin molecule to measure catalytic activity. 
Briefly, plants containing assembled immunoglobulin molecules were produced 
using the method and procedures described in Examples 1, 2, 3, 4, 5, 6 and 
8. The 6D4 immunoglobulin molecule was selected for expression in plants 
because a normally glycosylated 6D4 antibody produced in mice catalyzes 
the hydrolysis of carboxylic esters. See Tramontano et al., Science, 
234:1566 (1986). 
The 6D4 immunoglobulin was purified from the leaves of a tobacco plant 
expressing the immunoglobulin by sephacryl fractionation and absorption to 
Protein A-Sepharose. Briefly, midveins were removed from 10 grams (g) of 
young leaves which were then homogenized by hand in 50 ml of a 
homogenation buffer containing 50 mM of Tris-HCl at pH 8.0 and 1 mM PMSF. 
The resulting homogenate was centrifuged at 10,000 .times. g and the 
resulting supernatant concentrated to a final volume of 10 ml using a 
Centricon 30 (Amicon, Danvers, MA). The concentrated homogenate was then 
loaded onto a previously prepared sephacryl S-300 column. The column was 
eluted with 0.1M sodium acetate at pH 5.0 and 1 ml fractions of eluate 
collected. The amount of immunoglobulin present in each of the collected 
fractions was determined using the ELISA assay described in Example 4. 
The fractions containing the majority of the eluted immunoglobulin were 
pooled and extensively dialyzed against a binding buffer containing 1.5 M 
glycine at pH 8.9 and 3.0 M NaCl. After dialysis, the immunoglobulin was 
slowly passed twice over a column containing 2 g of protein A-sepharose 
(Pharmacia, Piscataway, N.J.) to allow the immunoglobulin to bind to the 
column. The protein A-sepharose was washed with 20 ml of binding buffer. 
The bound immunoglobulin was eluted with 10 ml of elution buffer 
containing 0.1M citrate at pH 6.0. The eluate was concentrated to 50 ug of 
immunoglobulin per ml using a Centricon 30. The concentrated 
immunoglobulin was then dialyzed against a 50 mM phosphate buffer at pH 
8.0. The final concentration of immunoglobulin present in the resulting 
solution was determined using an ELISA assay described in Example 8. 
The amino acid sequence of the resulting 6D4 immunoglobulin was determined 
using the methods described by Matsudaira, P., J. Biol. Chem., 
262:10035-10038 (1987). Briefly, the gamma heavy chain and kappa light 
chain of the 6D4 immunoglobulin were separated using sodium dodecyl 
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by loading 
approximately 1 ug of 6D4 immunoglobulin onto a 10% polyacrylamide gel. 
The immunoglobulin was electrophoresed until the gamma heavy chain and 
Kappa light chain were separated. The separated gamma heavy chain and 
kappa light chain were then blotted onto a polyvinylidene difluoride 
membranes as described by Matsudaira, P., J. Biol. Chem., 262:10035-10038 
(1987) and the amino acid sequence determined. 
Mouse derived 6D4 monoclonal antibody was purified from mouse ascites using 
the same procedure as that used to purify the antibody from plant leaves. 
Briefly, the mouse derived ascites fluid containing the 6D4 monoclonal 
antibody was fractionated on a Sephacryl (S-300) column and a protein 
A-sepharose column. The resulting purified mouse monoclonal 6D4 antibody 
was at a final concentration of 500 ug/ml in a 0.1M citrate, pH 6.0 
buffer. 
The plant derived and mouse derived 6D4 antibodies were assayed for 
catalytic activity by incubating the purified antibodies with a substrate 
(FIG. 9) in the presence of absence of a specific inhibitor (FIG. 9) as 
previously described by Tramontano et al., Science 234:1566-1569 (1986). 
Briefly, approximately 100 nM of mouse derived 6D4 antibody or plant 
derived 6D4 antibody was preincubated at 25 C. in 50 mM phosphate buffer 
at pH 8.0. A series of reaction admixtures were formed by admixing varying 
amounts of dioxane stock solution containing substrate to produce a series 
of reaction admixtures containing 5% dioxane and a substrate concentration 
ranging from 1 to 8 mM. The reaction admixtures were maintained for 1 hour 
at 25 C. and the hydrolysis of the ester substrate (FIG. 9) measured on a 
Hewlett-Packard 8452A diode array spectrophotometer by monitoring the 
adsorption change at 245 nanometers (nm). The maximum adsorption change 
was measured by adding a non-specific esterase (Sigma, St. Louis, Mo.) to 
a control reaction admixture. The kinetic parameters were obtained after 
subtraction of background hydrolysis, using the Lineweaver-Burke data 
treatment described by Tramontano et al., Science, 234:1566 (1986). The 
inhibition constants were determined by plotting the slopes obtained with 
both 100 nM and 300 nM phosphonate (Table 6). 
The catalytic activity of the purified plant derived and mouse derived 6D4 
antibodies as measured by K.sub.M, K.sub.1, V.sub.max and K.sub.cat is 
shown in Table 6. The plant derived and mouse derived 6D4 antibodies 
differed by less than one order of magnitude. 
TABLE 6 
______________________________________ 
Catalytic Activity of 6D4.sup.b. 
Source Tobacco Ascites 
______________________________________ 
.sup.K M 1.41 .times. 10.sup.-6 M 
9.8 .times. 10.sup.-6 M 
.sup.V max 
0.057 .times. 10.sup.-8 M sec.sup.-1 
0.31 .times. 10.sup.-8 M sec.sup.-1 
.sup.K 1 0.47 .times. 10.sup.-6 M 
1.06 .times. 10.sup.-6 M 
(competitive) (competitive) 
.sup.k cat 
0.008 sec.sup.-1 
0.025 sec.sup.-1 
______________________________________ 
.sup.b This data was analyzed using a linear regression. 
11. Production of Immunoglobulin with Heterologous Leader Sequences in 
Plants 
To determine the effects of a heterologous leader sequence on 
immunoglobulin assembly in plants, an immunoglobulin cDNA containing the 
signal and pre-sequence from the .alpha.-mating factor of Saccharomvces 
cereviseae in place of the native mouse leader sequences described in 
Example 1 was prepared. The sequence of the .alpha.-mating factor of 
Saccharomvces cereviseae is shown in Table 7 and has been described by 
Kurzan et al., Cell, 30:933-943 (1982). 
TABLE 7 
______________________________________ 
The sequence of the alpha-mating factor leader sequence 
coupled to either gamma or kappa chain 
GAATTCATTCAAGAATAGTTCAAACAAGAAGATTACA 
AACTATCAATTTCATACACAATATAAACGATTAAAAGA 
MRFPSIFTAVLFAASSAL 
AAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLP 
FSNSTNNGLLFINTTIASIAAKEEGVSLDLKR/DVVL 
(kappa chain); /EVEL (gamma chain) 
The underlined letters are the 5' untranslated nucleotides 
of the yeast pre-pro sequence. The remaining letters are 
the amino acids; / denotes the junction between the pre- 
pro sequence and the kappa or gamma chains. 
______________________________________ 
Briefly, the pre-pro sequence from the Saccharomvces cereviseae 
.alpha.-mating factor described by Kurzan et al., Cell. 30:933-943 (1982) 
was subcloned into M13mp18 by first isolating the Eco RI to Hind III 
restriction endonuclease fragment containing the .alpha.-mating factor 
from p69A. This .alpha.-mating factor containing restriction endonuclease 
fragment was then ligated to M13mp18 vector DNA that had been previously 
digested with Eco R1 and Hind III restriction endonucleases. The accuracy 
of this cloning step was determined by restriction endonuclease digestion 
of the resulting M13 clones containing the .alpha.-mating factor DNA. 
The 6D4 kappa and gamma chain vectors without endogenous mouse leader 
sequences prepared in Example 1, were digested with Hind III and the 
resulting 5' phosphate groups removed. The .alpha.-mating factor vector 
was digested with Hind III restriction endonuclease to produce a Hind III 
restriction endonuclease fragment containing the .alpha.-mating factor. 
The .alpha.-mating factor containing restriction endonuclease fragment was 
isolated using an electroeluter (BRL, Bethesda, Md.) after separation by 
agarose gel electrophoresis. 
The isolated .alpha.-mating factor containing restriction endonuclease 
fragment was ligated to the Hind III digested gamma and kappa vectors in 
separate ligation reactions. Oligonucleotide-directed mutagenesis was used 
to remove the surplus nucleotides between the end of the .alpha.-mating 
factor pre-pro sequence and the Gln codon (gamma chain) or the Asp codon 
(kappa chain) to produce chimeric cDNAs. The accuracy of the 
oligonucleotide-directed mutagenesis was confirmed by DNA sequencing. 
The chimeric cDNA's containing the .alpha.-mating factor pre-pro sequence 
and either the gamma or kappa immunoglobulin coding sequence were inserted 
into the PMON530 vector described by Rogers et al., Meth. Enzymol, 153:253 
(1987). Briefly, the chimeric cDNAs were attached to the pMON530 vector 
using the T4 DNA ligase. The products of the ligation reaction were 
introduced into E. coli using the bacterial strain and methods of Bethesda 
Research Laboratories (Bethesda, Md.). Individual plasmids (recombinant 
pMON530 containing the chimeric cDNA) were analyzed by restriction 
endonuclease digestion and sequencing. 
The resulting chimeric gamma and kappa cDNA expression vectors were used to 
transform leaf discs as described by Horsch et al., Science. 227:1229-1231 
(1985) and in Example 4. 
Individual plants expressing the gamma chain and individual regenerated 
plants expressing the kappa chain were selected. After confirming that the 
regenerates expressed either gamma or kappa chain using the ELISA 
described in Example 4, the individual regenerates were sexually crossed 
to produce a gamma X kappa progeny. These progeny were screen for antibody 
production using the ELISA assay described in Example 4. 
The individual regenerates expressing either the gamma.sub.mat chain or the 
kappa.sub.mat chain were crossed with plants expressing the native 6D4 
antibody containing the endogenous mouse leader peptide to produce progeny 
containing the native gamma chain and the kappa.sub.mat or progeny 
containing the gamma.sub.mat and the native kappa chain. These progeny 
were also screened using the ELISA assay described in Example 4. 
The level of antibody expression in each of these progeny was determined 
using the ELISA assay described in Example 4 and the results are reported 
in Table 8. 
TABLE 8 
______________________________________ 
Accumulation of Gamma or Kappa Chains 
and Antigen Binding of Gamma/Kappa Complexes. 
______________________________________ 
gamma mat.sup.a kappa mat 
743 .+-. 260 48 .+-. 8 
(1030) (72) 
gamma mat (kappa mat).sup.c 
kappa mat (gamma mat) 
2410 .+-. 1230 2280 .+-. 1300 
(7700) (7700) 
gamma mat (kappa mouse) 
kappa mat (gamma mouse) 
2615 .+-. 1505 2490 .+-. 1175 
(8300) (8300) 
gamma mat (kappa no leader) 
kappa mat (gamma no leader) 
705 .+-. 300 38 .+-. 8 
(0) (0) 
______________________________________ 
* Values are expressed in ng/mg total protein (mean .+-. S.E.) where 
purified 6D4 antibody from mouse ascites was used as the ELISA standard. 
Numbers in parenthesis are highest levels of expression. 
.sup.c .alpha.(K) refers to the abundance of .alpha. chain in a plant 
which also expresses K chain and vice versa (i.e. progeny of sexual cross 
as measured by ELISA. In these cases, values in parentheses are the resul 
of antibody binding to ELISA plates coated with the phosphonate antigen 
(P3) (previously described by Tramontano et al., Proc. Natl. Acad. Sci., 
USA, 83: 6736-6740 (1986)) conjugated to BSA Hiatt et al., Nature, 342: 
76-78 (1989). Only plants expressing the highest levels of K complex were 
used in the antigen binding assays. 
The individual gamma and kappa chains containing the Saccharomvces 
cereviseae leader sequence accumulated at nearly the same levels as 
constructs expressing the native mouse leader that were previously 
reported in Hiatt et al., Nature, 342:76-78 (1989). In addition, 
functional antibody was produced by crossing either gamma and kappa chains 
containing the same signal (gamma.sub.mat X kappa.sub.mat) or different 
signals (kappa.sub.mat X gamma.sub.native ; kappa.sub.native X 
gamma.sub.mat). This is in contrast to crosses of plants in which one 
parent expressed a immunoglobulin without a leader did not result in 
production of functional antibody molecules as reported by Hiatt et al., 
Nature, 342: 76-78 (1989). 
The fidelity of processing of the mouse immunoglobulin N-termini by the 
plant endomembrane system was determined by automated sequence analysis as 
described by p. Matsudaisa, J. Biol. Chem., 262:10035-10038 (1987). 
Mammalian kappa chains N terminal amino acid is typically aspartic acid as 
described by Kabat et al., Sequences of Proteins of Immunological 
Interest, Public Health Service, National Institutes of Health, Bethesda, 
Md. Many murine IgGI gamma chains are blocked by pyroglutanic as reported 
by Johnston et al., Bioch. Biophys. Res. Commun., 66: 843-847 (1975). 
Sequence analysis suggested that the gamma chains derived from plants 
expressing the native mouse leader contained a blocked N-terminus. The end 
terminal sequence of kappa chains expressing the native mouse leader was 
Asp-Val-Val-Leu indicating the appropriate proteolytic processing of the 
kappa chain. 
12. Glycosylation of Plant Derived Immunoglobulin Molecules 
To determine the gamma chain glycosylation pattern of the plant derived 
immunoglobulin, the purified antibody was blotted onto nitrocellulose and 
probed with biotinylated lectins as described by Faye et al., Anal. 
Biochem., 149:218-224 (1985). Briefly, the nitrocellulose membranes were 
incubated in a solution of 50 mM Tris-Cl, 0.5 m NaCl, 0.1 mM CaCl.sub.2, 
0.1 mM MgCl.sub.2, and 0.1 mM MnCl.sub.2 (TIBS) containing 1 ug/ml of a 
biotinylated lectin (Pierce, Rockford, Ill.) at room temperature for one 
hour. The filters were then washed with TIBS and incubated in TIBS 
containing 1 ug/ml streptavidin-alkaline phosphatase (Sigma, St. Louis, 
Mo.) for 1 hour at room temperature. The bound alkaline phosphatase was 
visualized using bromo-chloro-indolyl phosphate as described by Hiatt et 
al., Nature, 342:76-78 (1989). 
In some cases, the purified antibody was incubated with 40 milliunits of 
endoglycosidase H (Signal Chemical Co., St. Louis, MO) in 50 ul of 200 mM 
sodium acetate at pH 5.8 for 2 hours at 37C prior to blotting to remove 
high mannose type sugars. 
The results shown in FIG. 10 indicate that only Concanavalin A, specific 
for mannose and glucose bound to the plant-derived antibody whereas the 
mouse ascitesderived antibody was recognized by Concanavalin A as well as 
the lectins from the Ricinus communis, specific for terminal galactose 
residues (N-acetylgalactosamine), and to a lesser extent by wheat germ 
agglutinin that is specific for N-acetylglucosamine dimers having terminal 
sialic acid residues. The specificity of the various lectins is discussed 
in Kijimoto-Ochiai et al., Biochem. J., 257:43-49 (1989). The lectins from 
Datura stramonium specific for N-acetylglycosamine oligomers and N-acetyl 
lactosamine and the lectin from Phaseolus vulgaris that is specific for 
Gal .beta.1, 4 GlcNac .beta.1, 2 mannose, did not bind to either the plant 
or mouse ascites derived gamma chain. 
The elution of the lectins from the nitrocellulose blots using 
.alpha.-methylglucoside was used to compare the relative affinity of 
Concanavalin A binding to the plant-derived and mouse ascites derived 
antibodies as has been previously described by Johnston et al., Bioch. 
Biophys. Res. Commun., 66:843-847 (1975). Using this assay, the 
plant-derived and mouse ascites-derived antibodies are indistinguishable 
with regards to Concanavalin A affinity as well as the quantity of 
Concanavalin A binding per microgram of gamma chain. 
Digestion of either the plant-derived or mouse ascites-derived antibodies 
with endoglycosidase H using the conditions described by Trimvle et al., 
Anal. Biochem., 141:515-522 (1984) was carried out and the antibodies then 
transferred to nitrocellulose. The antibodies digested with 
endoglycosidase H displayed no reduction in Concanavalin A binding under 
conditions where Concanavalin A binding to ovalbumin was diminished. 
Taken together these results indicate that the plant-derived immunoglobulin 
is processed through similar cellular compartments as the mouse 
ascites-derived antibody. The gamma chain Concanavalin A binding and 
resistance of the glycan to digestion by endoglycosidase H as well as the 
correct kappa chain N-terminus indicate that the antibody is migrating 
from the endoplasmic reticulum to the Golgi and is being secreted through 
the plasma membrane as described by Walter et al., Annu. Rev. Cell. Biol., 
2:499-516 (1986) 
The differential binding of several of the lectins to the plant-derived 
antibody indicates that the final glycosylation pattern of the 
plant-derived antibody and the mouse ascites-derived antibody are 
different. The plant-derived antibody did not bind to the lectins specific 
for terminal galactose and terminal sialic acid whereas the mouse 
ascites-derived antibody did. 
13. Retention of Immunoglobulin Molecules Within the Plant Cell Wall 
The rate of secretion of immunoglobulins from plants protoplast that did 
not contain cell walls was compared to the rate of secretion of 
immunoglobulin from plant cells having intact cell walls. The preparation 
of protoplasts from plant cells has been described by Tricoli et al., 
Plant Cell Report, 5:334-337 (1986). Briefly, 1 cm.sup.2 pieces of tobacco 
leaf are incubated for 18 hours in a mixture of cellulysin (Calbiochem), 
macerase (Calbiochem) and driselase (Sigma) to digest cell walls and 
release protoplasts from the leaf. The protoplasts are purified by 
centrifugation (100 .times. g for 2 minutes) in 0.4 m Mannitol. 
The immunoglobulin produced by either protoplast or intact plant cells was 
labeled by resuspending 2.times.10.sup.6 protoplasts in 0.5 ml of a 
mannitol media containing and 10 uCi mCi of .sup.35 S-methionine. The 
cells were maintained in this labeling medium for 2 hours and an aliquot 
of cells and medium was removed to determine the incorporated of labeled 
methionine into the 6D4 antibody. The amount of labeled 6D4 antibody in 
the incubation media was determined by adhering the immunoglobulin 
contained in the medium to a protein-A Sepharose column and determining 
the total radioactive counts adhering to the column. The amount of labeled 
methionine incorporated in the cells into the 6D4 antibody was determined 
by preparing the cells and loading the lysate onto a 10% SDS-PAGE gel and 
electrophoresing the lysate for 2 hours, as previously described by Hiatt 
et al., J. Biol. Chem., 261:1293-1298 (1986). The region of the SDS-PAGE 
gel containing the 6D4 antibody was cut out and the labeled antibody 
eluded from the gel. The total amount of labeled antibody present was then 
determined. In addition, the same measurements was made after a further 
maintenance of 2 hours in the presence of 100 mM methionine. 
The callus cell lines were initiated over a period of 8 weeks by incubating 
leaf segments in the appropriate growth hormones as has been previously 
described by Hiatt et al., J. Biol. Chem., 261:1293-1298 (1986). The 
liquid suspensions cell lines were then initiated from clumps of the 
callus cells and used for the incorporation of .sup.35 S-methionine as 
described above. 
The results of this secretion analysis are shown in Table 9. After a 2 hour 
labeling period, a significant fraction of newly synthesized antibody was 
secreted from the protoplast. After a chase of 2 hours with 100 mM 
methionine, most of the total labeled antibody was secreted from the 
protoplast into the medium indicating that secretion of the antibody had 
occurred. In contrast, approximately 40% of the labeled antibody was 
retained within established callus suspension cell lines that had intact 
cell walls. These cells contain thin, primary cells walls and therefore 
retained the antibody within the cell wall. 
TABLE 9 
______________________________________ 
35 S-METHIONINE INCORPORATION INTO 6D4 AT 
2 HOURS (MEDIUM/CELLS) 
PROTOPLASTS PROTEIN A 0.33 
" SDS-PAGE 0.31 
CALLUS SUSPENSION CELLS 
PROTEIN A 0.39 
" SDS-PAGE 0.25 
INCORPORATION INTO 6D4 AFTER 2 HOUR CHASE 
PROTOPLASTS PROTEIN A 6.60 
" SDS-PAGE 6.31 
CALLUS SUSPENSION CELLS 
PROTEIN A 2.77 
" SDS-PAGE 2.14 
______________________________________ 
14. Production Of A Secretory IgA In A Plant Cell 
A. Isolation of Messenger RNA Coding for Pathogen Specific Variable Regions 
A secretory IgA immunospecific for a preselected antigen is produced in 
plant cells by first isolating the variable region coding genes from a 
preselected hybridoma. Messenger RNA is prepared according to the methods 
described by Chomczynski et al., Anal. Biochem., 162:156-159 (1987) using 
the manufacturers instructions and the RNA isolation kit produced by 
Stratagene (La Jolla, Calif.). Briefly, approximately 1.times.10.sup.7 
cells are homogenized in 10 ml of a denaturing solution containing 4.0M 
guanine isothiocyanate, 0.25M sodium citrate at pH 7.0, and 0.1M 
2-mercaptoethanol using a glass homogenizer. One ml of sodium acetate at a 
concentration of 2 M at pH 4.0 is admixed with the homogenized cells. One 
ml of water-saturated phenol is admixed to the denaturing solution 
containing the homogenized cells. Two ml of a chloroform: isoamyl alcohol 
(24:1 v/v) mixture is added to the homogenate. The homogenate is mixed 
vigorously for 10 seconds and is maintained on ice for 15 minutes. The 
homogenate is then transferred to a thick-walled 50 ml polypropylene 
centrifuge 2 (Fisher Scientific Company, Pittsburgh, Pa.). The solution is 
centrifuged at 10,000 .times. g for 20 minutes at 4C the upper 
RNA-containing aqueous layer is transferred to a fresh 50 ml polypropylene 
centrifuge 2 and is mixed with an equal volume of isopropyl alcohol. This 
solution is maintained at -20 C. for at least 1 hour to precipitate the 
RNA. The solution containing the precipitated RNA is centrifuged at 10,000 
.times. g for 20 minutes at 4 C. The pelleted total cellular RNA is 
collected and is dissolved in 3 ml of the denaturing solution described 
above. 
Three ml of the isopropyl alcohol is added to the resuspended total 
cellular RNA and is vigorously mixed. This solution is maintained at -20 C 
for at least 1 hour to precipitate the RNA. The solution containing the 
precipitated RNA is centrifuged at 10,000 .times. g for 10 minutes at 4 C. 
The pelleted RNA is washed once with a solution containing 75% ethanol. 
The pelleted RNA is dried under vacuum for 15 minutes and then is 
re-suspended in dimethyl pyrocarbonate treated (DEPC-H.sub.2 O) H.sub.2 O. 
The messenger RNA (mRNA) prepared above is enriched for sequences 
containing long poly A tracks as described in Molecular Cloning: A 
Laboratory Manual, Second Edition, Sambrook et al., eds., Cold Spring 
Harbor, N.Y. (1989). Briefly, one half of the total RNA isolated from the 
hybridoma cells is resuspended in 1 ml of DEPC-H.sub.2 O and is maintained 
at 65 C. for 5 minutes. One ml of 2.times.10 high salt loading buffer 
consisting of 100 mM Tris-Hcl, M sodium chloride, 2.0 mM disodium ethylene 
diamine tetraacetic acid (EDTA) at pH 7.5, and 0.2% sodium dodecyl 
sulphate (SDS) is added to the resuspended RNA and the mixture is allowed 
to cool to room temperature. The mixture is then applied to an oligo-dT 
(Collaborative Research Type 2 or Type 3) column that is previously 
prepared by washing the oligo-dT with a solution containing 0.1M sodium 
hydroxide and 5 mM EDTA and is then equilibrated in DEPC-H.sub.2 O. The 
column eluate is collected in a sterile polypropylene tube and is 
reapplied to the same column after heating the eluate for 5 minutes at 
65C. The oligo-dT column is then washed with 2 ml of high salt loading 
buffer consisting of 50 mM Tris-HCl at pH 7.5, 500 mM sodium chloride, 1 
mM EDTA at pH 7.5 and 0.1% SDS. The oligo-dT column is then washed with 2 
ml of 1 .times. medium salt buffer consisting of 50 mM Tris-HCl at pH 7.5, 
100 mM sodium chloride, 1 mM EDTA and 0.1% SDS. The messenger RNA is 
eluded from the oligo-dT column with 1 ml of buffer consisting of 10 mM 
Tris-HCl at pH 7.5, 1 mM EDTA at pH 7.5 and 0.05% SDS. The messenger RNA 
is purified by extracting this solution with a phenol/chloroform solution 
followed by a single extraction with 100% chloroform. The messenger RNA is 
concentrated by ethanol precipitation and then resuspended in DEPC-H.sub.2 
O and stored at -70 C. until used. 
The messenger RNA isolated by the above process contains messenger RNA 
coding for both the heavy and light chain variable regions that make up 
the antibody produced by the hybridoma. 
B. Isolation of the Variable Regions Using the Polymerase Chain Reaction 
In preparation for PCR amplification, the mRNA prepared according to the 
above examples is used as a template for cDNA synthesis by a primer 
extension reaction. In a typical 50 .mu.l transcription reaction, 5-10 
.mu.g of the hybridoma mRNA in water is first hybridized (annealed) with 
500 ng (50.0 pmol) of a 3' V.sub.H primer as described by Orlandi et al., 
Proc. Natl. Acad. Sci., U.S.A., 86:3833-3937 (1989 at 65C for 5 minutes. 
Subsequently the mixture is adjusted to 1.5 mM dATP, dCTP and dTTP, 40 mM 
Tris-HCl at pH 8.0, 8 mM MgCl.sub.2, 50 mM NaCl, and 2 mM spermidine. 
Moloney-Murine Leukemia Virus reverse transcriptase (26 units, Stratagene) 
is added to the solution and the solution is maintained for 1 hour at 37 
C. 
PCR amplification is performed in a 100 .mu.l reaction containing the 
products of the reverse transcription reaction (approximately 5 .mu.g of 
the cDNA/RNA hybrid), 300 ng of the 3' V" primer described by Orlandi et 
al., Proc. Natl. Acad. Sci., USA. 86:3833-3937 (1989). 300 ng each of the 
eight 5' V.sub.H primers also described by Orlandi et al., supra, 200 mM 
of a mixture of dNTP's, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 115 mM 
MgCl.sub.2 0.1% gelatin and 2 units of Taq DNA polymerase. The reaction 
mixture is overlaid with mineral oil and subjected to 40 cycles of 
amplification. Each amplification cycle involves a denaturation at 92 C. 
for 1 minute, annealing at 52 C. for 2 minutes and polynucleotide 
synthesis by primer extension (elongation) at 72 C. for 1.5 minutes. The 
amplified V.sub.H -coding DNA homolog containing samples are extracted 
twice with phenol-chloroform, once with chloroform, ethanol precipitated 
and are stored at -70 C. and 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. 
The light chain variable region is isolated in a similar fashion except 
that a 3' V.sub.H primer and a 5'V.sub.L primer specific for either the 
lambda or kappa light chain was used. The PCR amplification conditions 
were identical to those described for the heavy chain variable region 
isolation. 
C. Insertion Of The Pathogen Specific Heavy And Light Chain Variable Region 
Into A Plant Expression Vector 
The pathogen specific heavy and light chain variable regions are isolated 
as described above and are inserted into a plant expression vector 
containing the constant region of IgA. This vector is constructed using 
standard molecular biology techniques and is a derivative of pMON 530 that 
has both the immunoglobulin signal sequence from the 6D4 antibody as 
described in Example and the immunoglobulin alpha constant region isolated 
from MOPC 315 that has been fully sequenced and previously described by 
Auffray et al., Gene, 13:365-374 (1981). This vector also contains a 
polylinker region position between the immunoglobulin signal sequence and 
the IgA constant region gene to allow the pathogen specific heavy chain 
variable region to be easily inserted. The restriction endonuclease sites 
present in the polylinker are compatible with the restriction endonuclease 
sites present in the PCR primers used to isolate the heavy chain variable 
region. The pathogen specific heavy chain variable region is inserted into 
the vector by cutting the vector with the appropriate restriction enzymes 
and also cutting the pathogen specific variable region with the 
appropriate restriction enzymes sites that are present in the PCR primers 
used to isolate the variable. The pathogen specific variable region is 
then ligated into the vector. 
This vector is then introduced into a plant using the methods described in 
Example 4. Plants containing the pathogen specific IgA heavy chain are 
identified and then crossed with plants containing the pathogen specific 
light chain. 
Plants containing the pathogen specific light chain variable region coupled 
to an appropriate light chain are produced using similar techniques as the 
pathogen specific heavy chain variable region containing plants. 
A sexual cross is used to place the pathogen specific heavy and light 
chains in the same plant to produce a plant containing an assembled IgA. 
Plants containing the secretory component of IgA are produced by 
introducing the gene coding for the secretory component into a plant 
expression vector such as the pMON 530 vector. The sequence of the 
secretory component has been described by Mostov et al., Nature, 308:37 
(1984). The secretory component gene is inserted into the pMON 530 vector 
together with an appropriate signal sequence using standard molecular 
biology techniques. The resulting secretory component-containing vector is 
used to transform plant cells and produce plants containing and expressing 
the secretory component. 
Plants containing the J or joining chain of IgA immunoglobulin are produced 
by inserting the gene coding for the J chain into a plant expression 
vector as described for the secretory component, the light chain and heavy 
chain. The J chain gene has been sequenced by Max et al., J. Exp. Med., 
161:832-849 (1985). In addition, the sequence of other J chains is 
available in Sequences of Proteins of Immunological Interest, 4th edition, 
U.S. Dept. of Health and Human Services, (1987). This vector is used to 
produce plants expressing the J chain. 
These J chain expression plants are crossed with the plants expressing the 
secretory component to produce plants expressing both secretory component 
and J chain. These plants are then crossed with the plants expressing the 
pathogen-specific IgA antibody to produce plants expressing true secretory 
IgA that is made up of two IgA molecules, secretory component and J chain. 
D. Production Of Passive Immunity To A Selected Pathogen 
Plants producing secretory IgA were produced according to Example 11. These 
plants produced secretory IgA that was immunospecific for a Shigella 
toxin. This secretory IgA was produced by isolating the heavy and light 
chain variable regions from the hybridoma designated 13 C2 (ATCC 
#CRL1794). Plants expressing the secretory IgA contained approximately 1 
mg of secretory IgA for each 10 to 100 grams of plant material. These 
plants are harvested and used to produce passive immunity while the plant 
is still fresh. 
Adults in which passive immunity is desired are immunized by ingesting 10 
to 100 grams of plants expressing the secretory IgA, 1 to 4 times per day. 
This immunoglobulin ingestion is carried out for a total of 3 days and 
then the production of passive immunity is analyzed by ingesting a dose of 
bacteria containing Shigella toxin. The adults ingest approximately 
1.2.times.10.sup.9 colony-forming units of the Shigella bacteria suspended 
in 1 ounce of water containing sodium bicarbonate. Approximately 15 
minutes to 1/2 hour later the adults ingested 10 to 100 grams more of 
plant containing the secretory IgA. 
The adults are monitored for the presence of diarrhea for 1 to 2 days after 
ingesting the bacteria. The occurrence of diarrhea is greatly reduced in 
the adults ingesting the plant containing the secretory IgA as compared to 
other adults who did not ingest the secretory IgA-containing plant but 
were subjected to the same bacterial challenge. 
Plants containing a secretory IgA immunospecific for Shigella toxin and 
Shigella-like toxin (SLT1) are prepared by isolating the heavy and light 
chain variable regions from the hybridoma 13C2 (ATCC #CRL 1794). The 
plants contain approximately 1 mg of anti-Shigella antibody per 10 to 100 
grams of plant material. Plants containing the anti-Shigella antibody are 
isolated and homogenized and placed in an infant formula. 
Infants are given the equivalent of 6-600 mg of antibody present in the 
required amount of plant material daily in 3 or more doses as a supplement 
to their normal feeding. These infants are then followed to determine the 
incidence of Shigella disease in the infants after normal exposure to 
Shigella bacteria. Infants receiving the plant material containing the 
secretory IgA specific for Shigella toxin have a greatly reduced incidence 
of disease caused by Shigella when compared to infants exposed to the same 
amount of Shigella that did not receive the plant material containing the 
secretory IgA. 
The foregoing is intended as illustrative of the present invention but not 
limiting. Numerous variations and modifications can be effected without 
departing from the true spirit and scope of the invention.