DNA encoding human cystatin E

Disclosed is a human CysE polypeptide and DNA (RNA) encoding such polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for treating osteoporosis, tumor metastases, microbial infections, viral infection, septic shock, inflammation, retinal irritation, caries, cachexia and muscle wasting. Diagnostic methods for detecting mutations in the coding sequence and alterations in the concentration of the polypeptides in a sample derived from a host are also disclosed.

This invention relates to newly identified polynucleotides, polypeptides 
encoded by such polynucleotides, the use of such polynucleotides and 
polypeptides, as well as the production of such polynucleotides and 
polypeptides. More particularly, the polypeptide of the present invention 
has been putatively identified as human cystatin E, sometimes hereinafter 
referred to as "CysE". The invention also relates to inhibiting the action 
of such polypeptides. 
The cystatin superfamily comprises a group of cysteine proteinase 
inhibitors which are widely distributed in human tissues and body fluids, 
and which form tight and reversible complexes with cysteine proteinases 
such as cathepsins B, H, L, and S. The cystatins are most likely involved 
in the regulation of normal or pathological processes in which these 
proteinases participate. Thus, cystatins may influence the intra- and 
extracellular catabolism of proteins and peptides (Barret, A. J. and 
Kirchke, H., Methods Enzymol., 80:535-561 (1981)), regulate proteolytic 
processing of pro-hormones (Orlowski, M., Mol. Cell. Biochem., 52:49-74 
(1983)) and proenzymes (Taugner, R., et al., Histochemistry, 83:103-108 
(1985)), protect against penetration of normal tissues by malignant cells 
(Sloane, B. F., Semin. Cancer Biol., 1:137-152 (1990)) or microorganisms 
(Bjorck, L., et al., Nature, 337:385-386 (1989) and Bjorck, L., et al., J. 
Virol., 64:941-943 (1990)) and modulate local inflammatory processes in 
rheumatoid arthritis (Mort, J. S., et al., Arthritis Rheum., 27:509-515 
(1984)) and purulent bronchiectasis (Buttle, D. J., et al., Scand. J. 
Clin. Lab. Invest., 50:509-516 (1990)). 
The cystatin superfamily has been sub-divided into families I, II and III 
(also called the stefin, cystatin and kininogen families, respectively), 
each with members differing from those of the other families in structural 
organization and biological distribution (Barret, A. J., et al., Biochem. 
J., 236:312 (1986)). The family I cystatins A and B are small proteins 
consisting of single polypeptide chains of about 100 amino acid residues 
without disulfide bridges. The family II cystatins consist of polypeptide 
chains of approximately 120 amino acid residues with two intra-chain 
disulfide bonds. Finally, the family III cystatins, the kininogens, 
display a higher degree of structural complexity characterized by the 
presence of three family II cystatin-like domains, each with two disulfide 
bridges at positions homologous to those in family II cystatins 
(Muller-Esterl, W., et al., Transbiochem. Sci., 11:336-339 (1986)). Family 
I and II cystatins are mainly present intracellularly and in secretory 
fluids (Abrahamson, M., et al., J. Biol. Chem., 261:11282-11289 (1986)), 
whereas kinigogens are highly concentrated in blood plasma (Adam, A., et 
al., Clin. Chem., 31:423-426 (1985)). 
At least one type II cystatin, designated cystatin C, appears to be 
expressed in all tissues (Abrahamson, M., et al., Biochem. J., 268:287-294 
(1990)). In contrast, S-type cystatins are found predominantly in saliva 
(Abrahamson, M., et al., J. Biol. Chem., 261:11282-11289 (1986)). 
Cystatins and derivative peptides possess antibacterial and antiviral 
activities (Bjorck, et al. (1989, 1990)), consistent with their presence 
in secretions bathing epithelial surfaces directly exposed to the 
environment. The cystatins may also modulate the immune response. This 
could occur directly, by inhibiting cysteine proteases releases by 
macrophages (Bieth, J., Cysteine Proteinases and Their Inhibitors, V. 
Turk, ed. (Walter De Gruyter & Company, New York) pp. 693-703 (1986)), or 
indirectly, by inhibiting the chemotaxic response and the 
phagocytosis-associated respiratory burst of the cells (Leung-Tack, et 
al., Biol. Chem., 371:255-258 (1990)). This data suggests that type II 
cystatins might perform a variety of protective functions at epithelial 
surfaces. The human type II cystatin gene family consists of at least 
seven members. 
The disease hereditary cystatin C amyloid angiopathy (HCCAA) is associated 
with a Glu.fwdarw.Leu mutation in the gene encoding cystatin C. This leads 
to deposition of amyloid fibrils comprised of this mutant cystatin C in 
the cerebral arteries, which appears to cause fatal hemorrhaging (Ghiso, 
J., et al., PNAS. USA, 83:2974-2978 (1986)). 
The polypeptide of the present invention has been putatively identified as 
a CysE as a result of amino acid sequence homology to cystatin C. This 
identification has been made as a result of amino acid sequence homology. 
In accordance with one aspect of the present invention, there is provided a 
novel mature polypeptide, as well as biologically active and 
diagnostically or therapeutically useful fragments, analogs and 
derivatives thereof. The polypeptide of the present invention is of human 
origin. 
In accordance with another aspect of the present invention, there are 
provided isolated nucleic acid molecules encoding a polypeptide of the 
present invention, including mRNAs, DNAs, cDNAs, genomic DNAs as well as 
analogs and biologically active and diagnostically or therapeutically 
useful fragments thereof. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for producing such polypeptide by recombinant 
techniques comprising culturing recombinant prokaryotic and/or eukaryotic 
host cells, containing a human nucleic acid sequence encoding a 
polypeptide of the present invention, under conditions promoting 
expression of said protein and subsequent recovery of said protein. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for utilizing such polypeptide, or polynucleotide 
encoding such polypeptide for therapeutic purposes, for example, to 
inhibit cathepsins and prevent osteoporosis, tumor metastases, viral 
replication, inflammation, purulent bronchiectasis to protect the retina, 
to prevent leukoencephalopathy, to reduce caries, to treat allergic 
reactions, to treat cachexia and muscle wasting, and to prevent 
amyloidosis, and as a antimicrobial agent. 
In accordance with yet a further aspect of the present invention, there is 
also provided nucleic acid probes comprising nucleic acid molecules of 
sufficient length to specifically hybridize to nucleic acid sequences 
encoding a polypeptide of the present invention. 
In accordance with yet a further aspect of the present invention, there are 
provided antibodies against such polypeptides. 
In accordance with still another aspect of the present invention, there are 
provided diagnostic assays for detecting diseases or susceptibility to 
diseases related to mutations in the nucleic acid sequences encoding a 
polypeptide of the present invention. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for utilizing such polypeptides, or polynucleotides 
encoding such polypeptides, for in vitro purposes related to scientific 
research, for example, synthesis of DNA and manufacture of DNA vectors. 
These and other aspects of the present invention should be apparent to 
those skilled in the art from the teachings herein.

The polynucleotide of this invention was discovered in a cDNA library 
derived from primary culture amniotic cells. It is structurally related to 
the cystatin II superfamily. It contains an open reading frame encoding a 
protein of 149 amino acid residues of which approximately the first 28 
amino acids residues are the putative leader sequence such that the mature 
protein comprises 121 amino acids. The protein exhibits the highest degree 
of homology to human cystatin C with 37.273% identity and 49.091% 
similarity over a 110 amino acid stretch. 
The polynucleotide of the present invention may be in the form of RNA or in 
the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. 
The DNA may be double-stranded or single-stranded, and if single stranded 
may be the coding strand or non-coding (anti-sense) strand. The coding 
sequence which encodes the mature polypeptide may be identical to the 
coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of the deposited 
clone or may be a different coding sequence which coding sequence, as a 
result of the redundancy or degeneracy of the genetic code, encodes the 
same mature polypeptide as the DNA of FIG. 1 (SEQ ID NO:1) or the 
deposited cDNA. 
The polynucleotide which encodes for the mature polypeptide of FIG. 1 (SEQ 
ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may 
include, but is not limited to: only the coding sequence for the mature 
polypeptide; the coding sequence for the mature polypeptide and additional 
coding sequence such as a leader or secretory sequence or a proprotein 
sequence; the coding sequence for the mature polypeptide (and optionally 
additional coding sequence) and non-coding sequence, such as introns or 
non-coding sequence 5' and/or 3' of the coding sequence for the mature 
polypeptide. 
Thus, the term "polynucleotide encoding a polypeptide" encompasses a 
polynucleotide which includes only coding sequence for the polypeptide as 
well as a polynucleotide which includes additional coding and/or 
non-coding sequence. 
The present invention further relates to variants of the hereinabove 
described polynucleotides which encode for fragments, analogs and 
derivatives of the polypeptide having the deduced amino acid sequence of 
FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the 
deposited clone. The variant of the polynucleotide may be a naturally 
occurring allelic variant of the polynucleotide or a non-naturally 
occurring variant of the polynucleotide. 
Thus, the present invention includes polynucleotides encoding the same 
mature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same mature 
polypeptide encoded by the cDNA of the deposited clone as well as variants 
of such polynucleotides which variants encode for a fragment, derivative 
or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or the polypeptide 
encoded by the cDNA of the deposited clone. Such nucleotide variants 
include deletion variants, substitution variants and addition or insertion 
variants. 
As hereinabove indicated, the polynucleotide may have a coding sequence 
which is a naturally occurring allelic variant of the coding sequence 
shown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the deposited 
clone. As known in the art, an allelic variant is an alternate form of a 
polynucleotide sequence which may have a substitution, deletion or 
addition of one or more nucleotides, which does not substantially alter 
the function of the encoded polypeptide. 
The present invention also includes polynucleotides, wherein the coding 
sequence for the mature polypeptide may be fused in the same reading frame 
to a polynucleotide sequence which aids in expression and secretion of a 
polypeptide from a host cell, for example, a leader sequence which 
functions as a secretory sequence for controlling transport of a 
polypeptide from the cell. The polypeptide having a leader sequence is a 
preprotein and may have the leader sequence cleaved by the host cell to 
form the mature form of the polypeptide. The polynucleotides may also 
encode for a proprotein which is the mature protein plus additional 5' 
amino acid residues. A mature protein having a prosequence is a proprotein 
and is an inactive form of the protein. Once the prosequence is cleaved an 
active mature protein remains. 
Thus, for example, the polynucleotide of the present invention may encode 
for a mature protein, or for a protein having a prosequence or for a 
protein having both a prosequence and a presequence (leader sequence). 
The polynucleotides of the present invention may also have the coding 
sequence fused in frame to a marker sequence which allows for purification 
of the polypeptide of the present invention. The marker sequence may be a 
hexahistidine tag supplied by a pQE-9 vector to provide for purification 
of the mature polypeptide fused to the marker in the case of a bacterial 
host, or, for example, the marker sequence may be a hemagglutinin (HA) tag 
when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds 
to an epitope derived from the influenza hemagglutinin protein (Wilson, 
I., et al., Cell, 37:767 (1984)). 
The term "gene" means the segment of DNA involved in producing a 
polypeptide chain; it includes regions preceding and following the coding 
region (leader and trailer) as well as intervening sequences (introns) 
between individual coding segments (exons). 
Fragments of the full length CysE gene may be used as a hybridization probe 
for a cDNA library to isolate the full length CysE gene and to isolate 
other genes which have a high sequence similarity to the CysE gene or 
similar biological activity. Probes of this type preferably have at least 
30 bases and may contain, for example, 50 or more bases. The probe may 
also be used to identify a cDNA clone corresponding to a full length 
transcript and a genomic clone or clones that contain the complete CysE 
gene including regulatory and promotor regions, exons, and introns. An 
example of a screen comprises isolating the coding region of the CysE gene 
by using the known DNA sequence to synthesize an oligonucleotide probe. 
Labeled oligonucleotides having a sequence complementary to that of the 
gene of the present invention are used to screen a library of human cDNA, 
genomic DNA or mRNA to determine which members of the library the probe 
hybridizes to. 
The present invention further relates to polynucleotides which hybridize to 
the hereinabove-described sequences if there is at least 70%, preferably 
at least 90%, and more preferably at least 95% identity between the 
sequences. The present invention particularly relates to polynucleotides 
which hybridize under stringent conditions to the hereinabove-described 
polynucleotides. As herein used, the term "stringent conditions" means 
hybridization will occur only if there is at least 95% and preferably at 
least 97% identity between the sequences. The polynucleotides which 
hybridize to the hereinabove described polynucleotides in a preferred 
embodiment encode polypeptides which either retain substantially the same 
biological function or activity as the mature polypeptide encoded by the 
cDNAs of FIG. 1 (SEQ ID NO:1) or the deposited cDNA(s). 
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 
bases, and more preferably at least 50 bases which hybridize to a 
polynucleotide of the present invention and which has an identity thereto, 
as hereinabove described, and which may or may not retain activity. For 
example, such polynucleotides may be employed as probes for the 
polynucleotide of SEQ ID NO:1, for example, for recovery of the 
polynucleotide or as a diagnostic probe or as a PCR primer. 
Thus, the present invention is directed to polynucleotides having at least 
a 70% identity, preferably at least 90% and more preferably at least a 95% 
identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 
as well as fragments thereof, which fragments have at least 30 bases and 
preferably at least 50 bases and to polypeptides encoded by such 
polynucleotides. 
The deposit(s) referred to herein will be maintained under the terms of the 
Budapest Treaty on the International Recognition of the Deposit of 
Micro-organisms for purposes of Patent Procedure. These deposits are 
provided merely as convenience to those of skill in the art and are not an 
admission that a deposit is required under 35 U.S.C. .sctn.112. The 
sequence of the polynucleotides contained in the deposited materials, as 
well as the amino acid sequence of the polypeptides encoded thereby, are 
incorporated herein by reference and are controlling in the event of any 
conflict with any description of sequences herein. A license may be 
required to make, use or sell the deposited materials, and no such license 
is hereby granted. 
The present invention further relates to a CysE polypeptide which has the 
deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or which has the amino 
acid sequence encoded by the deposited cDNA, as well as fragments, analogs 
and derivatives of such polypeptide. 
The terms "fragment," "derivative" and "analog" when referring to the 
polypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the deposited cDNA, 
means a polypeptide which retains essentially the same biological function 
or activity as such polypeptide. Thus, an analog includes a proprotein 
which can be activated by cleavage of the proprotein portion to produce an 
active mature polypeptide. 
The polypeptide of the present invention may be a recombinant polypeptide, 
a natural polypeptide or a synthetic polypeptide, preferably a recombinant 
polypeptide. 
The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ ID 
NO:2) or that encoded by the deposited cDNA may be (i) one in which one or 
more of the amino acid residues are substituted with a conserved or 
non-conserved amino acid residue (preferably a conserved amino acid 
residue) and such substituted amino acid residue may or may not be one 
encoded by the genetic code, or (ii) one in which one or more of the amino 
acid residues includes a substituent group, or (iii) one in which the 
mature polypeptide is fused with another compound, such as a compound to 
increase the half-life of the polypeptide (for example, polyethylene 
glycol), or (iv) one in which the additional amino acids are fused to the 
mature polypeptide, such as a leader or secretory sequence or a sequence 
which is employed for purification of the mature polypeptide or a 
proprotein sequence. Such fragments, derivatives and analogs are deemed to 
be within the scope of those skilled in the art from the teachings herein. 
The polypeptides and polynucleotides of the present invention are 
preferably provided in an isolated form, and preferably are purified to 
homogeneity. 
The term "isolated" means that the material is removed from its original 
environment (e.g., the natural environment if it is naturally occurring). 
For example, a naturally-occurring polynucleotide or polypeptide present 
in a living animal is not isolated, but the same polynucleotide or 
polypeptide, separated from some or all of the coexisting materials in the 
natural system, is isolated. Such polynucleotides could be part of a 
vector and/or such polynucleotides or polypeptides could be part of a 
composition, and still be isolated in that such vector or composition is 
not part of its natural environment. 
The polypeptides of the present invention include the polypeptide of SEQ ID 
NO:2 (in particular the mature polypeptide) as well as polypeptides which 
have at least 70% similarity (preferably at least 70% identity) to the 
polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity 
(more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 
and still more preferably at least 95% similarity (still more preferably 
at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include 
portions of such polypeptides with such portion of the polypeptide 
generally containing at least 30 amino acids and more preferably at least 
50 amino acids. 
As known in the art "similarity" between two polypeptides is determined by 
comparing the amino acid sequence and its conserved amino acid substitutes 
of one polypeptide to the sequence of a second polypeptide. 
Fragments or portions of the polypeptides of the present invention may be 
employed for producing the corresponding full-length polypeptide by 
peptide synthesis; therefore, the fragments may be employed as 
intermediates for producing the full-length polypeptides. Fragments or 
portions of the polynucleotides of the present invention may be used to 
synthesize full-length polynucleotides of the present invention. 
The present invention also relates to vectors which include polynucleotides 
of the present invention, host cells which are genetically engineered with 
vectors of the invention and the production of polypeptides of the 
invention by recombinant techniques. 
Host cells are genetically engineered (transduced or transformed or 
transfected) with the vectors of this invention which may be, for example, 
a cloning vector or an expression vector. The vector may be, for example, 
in the form of a plasmid, a viral particle, a phage, etc. The engineered 
host cells can be cultured in conventional nutrient media modified as 
appropriate for activating promoters, selecting transformants or 
amplifying the CysE genes. The culture conditions, such as temperature, pH 
and the like, are those previously used with the host cell selected for 
expression, and will be apparent to the ordinarily skilled artisan. 
The polynucleotides of the present invention may be employed for producing 
polypeptides by recombinant techniques. Thus, for example, the 
polynucleotide may be included in any one of a variety of expression 
vectors for expressing a polypeptide. Such vectors include chromosomal, 
nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; 
bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors 
derived from combinations of plasmids and phage DNA, viral DNA such as 
vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other 
vector may be used as long as it is replicable and viable in the host. 
The appropriate DNA sequence may be inserted into the vector by a variety 
of procedures. In general, the DNA sequence is inserted into an 
appropriate restriction endonuclease site(s) by procedures known in the 
art. Such procedures and others are deemed to be within the scope of those 
skilled in the art. 
The DNA sequence in the expression vector is operatively linked to an 
appropriate expression control sequence(s) (promoter) to direct mRNA 
synthesis. As representative examples of such promoters, there may be 
mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda 
P.sub.L promoter and other promoters known to control expression of genes 
in prokaryotic or eukaryotic cells or their viruses. The expression vector 
also contains a ribosome binding site for translation initiation and a 
transcription terminator. The vector may also include appropriate 
sequences for amplifying expression. 
In addition, the expression vectors preferably contain one or more 
selectable marker genes to provide a phenotypic trait for selection of 
transformed host cells such as dihydrofolate reductase or neomycin 
resistance for eukaryotic cell culture, or such as tetracycline or 
ampicillin resistance in E. coli. 
The vector containing the appropriate DNA sequence as hereinabove 
described, as well as an appropriate promoter or control sequence, may be 
employed to transform an appropriate host to permit the host to express 
the protein. 
As representative examples of appropriate hosts, there may be mentioned: 
bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; 
fungal cells, such as yeast; insect cells such as Drosophila S2 and 
Spodoptera Sf 9; animal cells such as CHO, COS or Bowes melanoma; 
adenoviruses; plant cells, etc. The selection of an appropriate host is 
deemed to be within the scope of those skilled in the art from the 
teachings herein. 
More particularly, the present invention also includes recombinant 
constructs comprising one or more of the sequences as broadly described 
above. The constructs comprise a vector, such as a plasmid or viral 
vector, into which a sequence of the invention has been inserted, in a 
forward or reverse orientation. In a preferred aspect of this embodiment, 
the construct further comprises regulatory sequences, including, for 
example, a promoter, operably linked to the sequence. Large numbers of 
suitable vectors and promoters are known to those of skill in the art, and 
are commercially available. The following vectors are provided by way of 
example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, 
psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A 
(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); 
Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, PBPV, 
PMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used 
as long as they are replicable and viable in the host. 
Promoter regions can be selected from any desired gene using CAT 
(chloramphenicol transferase) vectors or other vectors with selectable 
markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named 
bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, 
P.sub.L and trp. Eukaryotic promoters include CMV immediate early, HSV 
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse 
metallothionein-I. Selection of the appropriate vector and promoter is 
well within the level of ordinary skill in the art. 
In a further embodiment, the present invention relates to host cells 
containing the above-described constructs. The host cell can be a higher 
eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, 
such as a yeast cell, or the host cell can be a prokaryotic cell, such as 
a bacterial cell. Introduction of the construct into the host cell can be 
effected by calcium phosphate transfection, DEAE-Dextran mediated 
transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic 
Methods in Molecular Biology, (1986)). 
The constructs in host cells can be used in a conventional manner to 
produce the gene product encoded by the recombinant sequence. 
Alternatively, the polypeptides of the invention can be synthetically 
produced by conventional peptide synthesizers. 
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or 
other cells under the control of appropriate promoters. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the present invention. Appropriate 
cloning and expression vectors for use with prokaryotic and eukaryotic 
hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory 
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure 
of which is hereby incorporated by reference. 
Transcription of the DNA encoding the polypeptides of the present invention 
by higher eukaryotes is increased by inserting an enhancer sequence into 
the vector. Enhancers are cis-acting elements of DNA, usually about from 
10 to 300 bp that act on a promoter to increase its transcription. 
Examples include the SV40 enhancer on the late side of the replication 
origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the 
polyoma enhancer on the late side of the replication origin, and 
adenovirus enhancers. 
Generally, recombinant expression vectors will include origins of 
replication and selectable markers permitting transformation of the host 
cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae 
TRP1 gene, and a promoter derived from a highly-expressed gene to direct 
transcription of a downstream structural sequence. Such promoters can be 
derived from operons encoding glycolytic enzymes such as 
3-phosphoglycerate kinase (PGK), .alpha.-factor, acid phosphatase, or heat 
shock proteins, among others. The heterologous structural sequence is 
assembled in appropriate phase with translation initiation and termination 
sequences, and preferably, a leader sequence capable of directing 
secretion of translated protein into the periplasmic space or 
extracellular medium. Optionally, the heterologous sequence can encode a 
fusion protein including an N-terminal identification peptide imparting 
desired characteristics, e.g., stabilization or simplified purification of 
expressed recombinant product. 
Useful expression vectors for bacterial use are constructed by inserting a 
structural DNA sequence encoding a desired protein together with suitable 
translation initiation and termination signals in operable reading phase 
with a functional promoter. The vector will comprise one or more 
phenotypic selectable markers and an origin of replication to ensure 
maintenance of the vector and to, if desirable, provide amplification 
within the host. Suitable prokaryotic hosts for transformation include E. 
coli, Bacillus subtilis, Salmonella typhimurium and various species within 
the genera Pseudomonas, Streptomyces, and Staphylococcus, although others 
may also be employed as a matter of choice. 
As a representative but nonlimiting example, useful expression vectors for 
bacterial use can comprise a selectable marker and bacterial origin of 
replication derived from commercially available plasmids comprising 
genetic elements of the well known cloning vector pBR322 (ATCC 37017). 
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine 
Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). 
These pBR322 "backbone" sections are combined with an appropriate promoter 
and the structural sequence to be expressed. 
Following transformation of a suitable host strain and growth of the host 
strain to an appropriate cell density, the selected promoter is induced by 
appropriate means (e.g., temperature shift or chemical induction) and 
cells are cultured for an additional period. 
Cells are typically harvested by centrifugation, disrupted by physical or 
chemical means, and the resulting crude extract retained for further 
purification. 
Microbial cells employed in expression of proteins can be disrupted by any 
convenient method, including freeze-thaw cycling, sonication, mechanical 
disruption, or use of cell lysing agents, such methods are well known to 
those skilled in the art. 
Various mammalian cell culture systems can also be employed to express 
recombinant protein. Examples of mammalian expression systems include the 
COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 
23:175 (1981), and other cell lines capable of expressing a compatible 
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. 
Mammalian expression vectors will comprise an origin of replication, a 
suitable promoter and enhancer, and also any necessary ribosome binding 
sites, polyadenylation site, splice donor and acceptor sites, 
transcriptional termination sequences, and 5' flanking nontranscribed 
sequences. DNA sequences derived from the SV40 splice, and polyadenylation 
sites may be used to provide the required nontranscribed genetic elements. 
The the polypeptide of the present invention can be recovered and purified 
from recombinant cell cultures by methods including ammonium sulfate or 
ethanol precipitation, acid extraction, anion or cation exchange 
chromatography, phosphocellulose chromatography, hydrophobic interaction 
chromatography, affinity chromatography, hydroxylapatite chromatography 
and lectin chromatography. Protein refolding steps can be used, as 
necessary, in completing configuration of the mature protein. Finally, 
high performance liquid chromatography (HPLC) can be employed for final 
purification steps. 
The polypeptides of the present invention may be a naturally purified 
product, or a product of chemical synthetic procedures, or produced by 
recombinant techniques from a prokaryotic or eukaryotic host (for example, 
by bacterial, yeast, higher plant, insect and mammalian cells in culture). 
Depending upon the host employed in a recombinant production procedure, 
the polypeptides of the present invention may be glycosylated or may be 
non-glycosylated. Polypeptides of the invention may also include an 
initial methionine amino acid residue. 
The CysE polypeptide of the present invention may be employed to inhibit 
human cathepsin enzymes and the resulting pathologies related to the 
action of these cathepsins. For example, CysE may be employed to treat 
osteoporosis, behcet disease, hypercalcemia, osteomalicia, allergic skin 
diseases, allergic rhinitis and allergic purpura. 
It is also thought that the cathepsins play a vital role in the 
metastasizing of tumors and, accordingly, CysE may be employed to prevent 
tumor metastases. 
The CysE polypeptide may be employed as an antimicrobial agent to halt the 
growth of certain microbial agents, for example, streptococci and to 
reduce dental caries by reducing the production of acids which contribute 
to caries. 
The CysE polypeptide may also be employed as an antiviral agent to treat 
infection caused by viruses, for example, to prevent the replication of 
herpes simplex virus (HSV). The CysE polypeptide may also be employed to 
protect the retina against attack by the cystein proteinases. 
The CysE polypeptide of the present invention may also be employed to treat 
cachexia and muscle wasting by preventing the action of cysteine 
proteinases. 
The CysE polypeptide may also be employed to modify inflammation, for 
example, that associated with rheumatoid arthritis, and to treat septic 
shock. The CysE polypeptide may also be employed to treat purulent 
bronchiectasis. 
The polynucleotides and polypeptides of the present invention may be 
employed as research reagents and materials for discovery of treatments 
and diagnostics to human disease. 
This invention provides a method for identification of the receptor for the 
cystatin E polypeptide. The gene encoding the receptor can be identified 
by numerous methods known to those of skill in the art, for example, 
ligand panning and FACS sorting (Coligan, et al., Current Protocols in 
Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning is 
employed wherein polyadenylated RNA is prepared from a cell responsive to 
the cystatin E polypeptide, and a cDNA library created from this RNA is 
divided into pools and used to transfect COS cells or other cells that are 
not responsive to the cystatin E polypeptide. Transfected cells which are 
grown on glass slides are exposed to labeled cystatin E polypeptide. The 
cystatin E polypeptide can be labeled by a variety of means including 
iodination or inclusion of a recognition site for a site-specific protein 
kinase. Following fixation and incubation, the slides are subjected to 
auto-radiographic analysis. Positive pools are identified and sub-pools 
are prepared and re-transfected using an iterative sub-pooling and 
re-screening process, eventually yielding a single clone that encodes the 
putative receptor. As an alternative approach for receptor identification, 
labeled ligand can be photoaffinity linked with cell membrane or extract 
preparations that express the receptor molecule. Cross-linked material is 
resolved by PAGE and exposed to X-ray film. The labeled complex containing 
the ligand-receptor can be excised, resolved into peptide fragments, and 
subjected to protein microsequencing. The amino acid sequence obtained 
from microsequencing would be used to design a set of degenerate 
oligonucleotide probes to screen a cDNA library to identify the gene 
encoding the putative receptor. 
This invention provides a method of screening compounds to identify those 
which bind to the cystatin E receptor and induce a second messenger 
response therefrom. As an example, a mammalian cell or membrane 
preparation expressing the cystatin E receptor is incubated in the 
presence of the compound to be screened. The response of a known second 
messenger system following interaction of the compound and the receptor is 
measured and compared to the second messenger response induced by cystatin 
E. Such second messenger systems include but are not limited to, cAMP 
guanylate cyclase, ion channels or phosphoinositide hydrolysis. 
The polypeptide of the present invention and agonist compounds may be 
assayed for an ability to inhibit cyteine proteinase activity which assay 
comprises determining equilibrium constants for dissociation (K.sub.i) of 
cystatin E complexes with papain and human cathepsin B, by continuous rate 
assays with 10 .mu.M Z-Phe-Arg-NHMec as substrate in 100 M sodium 
phosphate buffer (Nicklin, M. J. H., and Barrett, A. J., Biochem. J., 
223:245-253 (1984). The buffer contains 1 mM dithiothreitol and 2 mM EDTA 
and is adjusted to pH 6.5 for papain assay and to pH 6.0 for cathepsin B 
assays. Cathepsin B is preincubated for 20 min in assay buffer at room 
temperature before use. The enzyme concentrations in the assays are 
0.05-0.25 nM. The highest cystatin E concentration tried in cathepsin B 
assays is 100 nM. The inhibitor concentrations giving informative 
inhibition, i.e., resulting in a new steady state rate within 1 hour after 
addition of inhibitor, are 20-50 nM in the papain assays. Substrate 
hydrolysis at 37.degree. C. is monitored in a Perkin-Elmer Cetus LS50 
fluorometer at excitation and emission wavelengths of 360 and 460 nm, 
respectively. K.sub.m values for hydrolysis of Z-Phe-Arg-NHMec under the 
assay are used to compensate obtained apparent K.sub.i values for 
substrate induced dissociation of inhibitor, by the relationship: Apparent 
K.sub.i =K.sub.i (1+[S]/K.sub.m). 
The polypeptides of the present invention and agonist compounds may be 
employed in combination with a suitable pharmaceutical carrier. Such 
compositions comprise a therapeutically effective amount of the 
polypeptide or agonist, and a pharmaceutically acceptable carrier or 
excipient. Such a carrier includes but is not limited to saline, buffered 
saline, dextrose, water, glycerol, ethanol, and combinations thereof. The 
formulation should suit the mode of administration. 
The invention also provides a pharmaceutical pack or kit comprising one or 
more containers filled with one or more of the ingredients of the 
pharmaceutical compositions of the invention. Associated with such 
container(s) can be a notice in the form prescribed by a governmental 
agency regulating the manufacture, use or sale of pharmaceuticals or 
biological products, which notice reflects approval by the agency of 
manufacture, use or sale for human administration. In addition, the 
polypeptides or agonists of the present invention may be employed in 
conjunction with other therapeutic compounds. 
The pharmaceutical compositions may be administered in a convenient manner 
such as by the oral, topical, intravenous, intraperitoneal, intramuscular, 
subcutaneous, intranasal or intradermal routes. The pharmaceutical 
compositions are administered in an amount which is effective for treating 
and/or prophylaxis of the specific indication. In general, they are 
administered in an amount of at least about 10 .mu.g/kg body weight and in 
most cases they will be administered in an amount not in excess of about 8 
mg/Kg body weight per day. In most cases, the dosage is from about 10 
.mu.g/kg to about 1 mg/kg body weight daily, taking into account the 
routes of administration, symptoms, etc. 
The CysE polypeptides and agonist compounds which are polypeptides may also 
be employed in accordance with the present invention by expression of such 
polypeptides in vivo, which is often referred to as "gene therapy." 
Thus, for example, cells from a patient may be engineered with a 
polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the 
engineered cells then being provided to a patient to be treated with the 
polypeptide. Such methods are well-known in the art and are apparent from 
the teachings herein. For example, cells may be engineered by the use of a 
retroviral plasmid vector containing RNA encoding a polypeptide of the 
present invention. 
Similarly, cells may be engineered in vivo for expression of a polypeptide 
in vivo by, for example, procedures known in the art. For example, a 
packaging cell is transduced with a retroviral plasmid vector containing 
RNA encoding a polypeptide of the present invention such that the 
packaging cell now produces infectious viral particles containing the gene 
of interest. These producer cells may be administered to a patient for 
engineering cells in vivo and expression of the polypeptide in vivo. These 
and other methods for administering a polypeptide of the present invention 
by such method should be apparent to those skilled in the art from the 
teachings of the present invention. 
Retroviruses from which the retroviral plasmid vectors hereinabove 
mentioned may be derived include, but are not limited to, Moloney Murine 
Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma 
Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia 
virus, human immunodeficiency virus, adenovirus, Myeloproliferative 
Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral 
plasmid vector is derived from Moloney Murine Leukemia Virus. 
The vector includes one or more promoters. Suitable promoters which may be 
employed include, but are not limited to, the retroviral LTR; the SV40 
promoter; and the human cytomegalovirus (CMV) promoter described in 
Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other 
promoter (e.g., cellular promoters such as eukaryotic cellular promoters 
including, but not limited to, the histone, pol III, and .beta.-actin 
promoters). Other viral promoters which may be employed include, but are 
not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and 
B19 parvovirus promoters. The selection of a suitable promoter will be 
apparent to those skilled in the art from the teachings contained herein. 
The nucleic acid sequence encoding the polypeptide of the present invention 
is under the control of a suitable promoter. Suitable promoters which may 
be employed include, but are not limited to, adenoviral promoters, such as 
the adenoviral major late promoter; or hetorologous promoters, such as the 
cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) 
promoter; inducible promoters, such as the MMT promoter, the 
metallothionein promoter; heat shock promoters; the albumin promoter; the 
ApoAI promoter; human globin promoters; viral thymidine kinase promoters, 
such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs 
(including the modified retroviral LTRs hereinabove described); the 
.beta.-actin promoter; and human growth hormone promoters. The promoter 
also may be the native promoter which controls the gene encoding the 
polypeptide. 
The retroviral plasmid vector is employed to transduce packaging cell lines 
to form producer cell lines. Examples of packaging cells which may be 
transfected include, but are not limited to, the PE501, 17, .psi.-2, 
.psi.-AM, 2, T19-14X, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, 
GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 
Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its 
entirety. The vector may transduce the packaging cells through any means 
known in the art. Such means include, but are not limited to, 
electroporation, the use of liposomes, and CaPO.sub.4 precipitation. In 
one alternative, the retroviral plasmid vector may be encapsulated into a 
liposome, or coupled to a lipid, and then administered to a host. 
The producer cell line generates infectious retroviral vector particles 
which include the nucleic acid sequence(s) encoding the polypeptides. Such 
retroviral vector particles then may be employed, to transduce eukaryotic 
cells, either in vitro or in vivo. The transduced eukaryotic cells will 
express the nucleic acid sequence(s) encoding the polypeptide. Bukaryotic 
cells which may be transduced include, but are not limited to, embryonic 
stem cells, embryonic carcinoma cells, as well as hematopoietic stem 
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial 
cells, and bronchial epithelial cells. 
The disease hereditary cystatin C amaloid angiopathy causes fatal 
hemorrhaging, and may be associated with Alzheimer's disease, Down's 
syndrome, Parkinson's, dimentia, and could lead to death before age 40. 
This invention, therefore, relates to the use of the CysE gene as a 
diagnostic. Detection of a mutated form of CysE will allow a diagnosis of 
a disease similar to HCCAA which results from a mutation in the CysE gene. 
Individuals carrying mutations in the human CysE gene may be detected at 
the DNA level by a variety of techniques. Nucleic acids for diagnosis may 
be obtained from a patient's cells, including but not limited to blood, 
urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be 
used directly for detection or may be amplified enzymatically by using PCR 
(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA or cDNA 
may also be used for the same purpose. As an example, PCR primers 
complementary to the nucleic acid encoding CysE can be used to identify 
and analyze CysE mutations. For example, deletions and insertions can be 
detected by a change in size of the amplified product in comparison to the 
normal genotype. Point mutations can be identified by hybridizing 
amplified DNA to radiolabeled CysE RNA or alternatively, radiolabeled CysE 
antisense DNA sequences. Perfectly matched sequences can be distinguished 
from mismatched duplexes by RNase A digestion or by differences in melting 
temperatures. 
Sequence differences between the reference gene and genes having mutations 
may be revealed by the direct DNA sequencing method. In addition, cloned 
DNA segments may be employed as probes to detect specific DNA segments. 
The sensitivity of this method is greatly enhanced when combined with PCR. 
For example, a sequencing primer is used with double-stranded PCR product 
or a single-stranded template molecule generated by a modified PCR. The 
sequence determination is performed by conventional procedures with 
radiolabeled nucleotide or by automatic sequencing procedures with 
fluorescent-tags. 
Genetic testing based on DNA sequence differences may be achieved by 
detection of alteration in electrophoretic mobility of DNA fragments in 
gels with or without denaturing agents. Small sequence deletions and 
insertions can be visualized by high resolution gel electrophoresis. DNA 
fragments of different sequences may be distinguished on denaturing 
formamide gradient gels in which the mobilities of different DNA fragments 
are retarded in the gel at different positions according to their specific 
melting or partial melting temperatures (see, e.g., Myers et al., Science, 
230:1242 (1985)). 
Sequence changes at specific locations may also be revealed by nuclease 
protection assays, such as RNase and S1 protection or the chemical 
cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)). 
Thus, the detection of a specific DNA sequence may be achieved by methods 
such as hybridization, RNase protection, chemical cleavage, direct DNA 
sequencing or the use of restriction enzymes, (e.g., Restriction Fragment 
Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA. 
In addition to more conventional gel-electrophoresis and DNA sequencing, 
mutations can also be detected by in situ analysis. 
The sequences of the present invention are also valuable for chromosome 
identification. The sequence is specifically targeted to and can hybridize 
with a particular location on an individual human chromosome. Moreover, 
there is a current need for identifying particular sites on the 
chromosome. Few chromosome marking reagents based on actual sequence data 
(repeat polymorphisms) are presently available for marking chromosomal 
location. The mapping of DNAs to chromosomes according to the present 
invention is an important first step in correlating those sequences with 
genes associated with disease. 
Briefly, sequences can be mapped to chromosomes by preparing PCR primers 
(preferably 15-25 bp) from the cDNA. Computer analysis of the 3' 
untranslated region of the gene is used to rapidly select primers that do 
not span more than one exon in the genomic DNA, thus complicating the 
amplification process. These primers are then used for PCR screening of 
somatic cell hybrids containing individual human chromosomes. Only those 
hybrids containing the human gene corresponding to the primer will yield 
an amplified fragment. 
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a 
particular DNA to a particular chromosome. Using the present invention 
with the same oligonucleotide primers, sublocalization can be achieved 
with panels of fragments from specific chromosomes or pools of large 
genomic clones in an analogous manner. Other mapping strategies that can 
similarly be used to map to its chromosome include in situ hybridization, 
prescreening with labeled flow-sorted chromosomes and preselection by 
hybridization to construct chromosome specific-cDNA libraries. 
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase 
chromosomal spread can be used to provide a precise chromosomal location 
in one step. This technique can be used with cDNA having at least 50 or 60 
bases. For a review of this technique, see Verma et al., Human 
Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York 
(1988). 
Once a sequence has been mapped to a precise chromosomal location, the 
physical position of the sequence on the chromosome can be correlated with 
genetic map data. Such data are found, for example, in V. McKusick, 
Mendelian Inheritance in Man (available on line through Johns Hopkins 
University Welch Medical Library). The relationship between genes and 
diseases that have been mapped to the same chromosomal region are then 
identified through linkage analysis (coinheritance of physically adjacent 
genes). 
Next, it is necessary to determine the differences in the cDNA or genomic 
sequence between affected and unaffected individuals. If a mutation is 
observed in some or all of the affected individuals but not in any normal 
individuals, then the mutation is likely to be the causative agent of the 
disease. 
With current resolution of physical mapping and genetic mapping techniques, 
a cDNA precisely localized to a chromosomal region associated with the 
disease could be one of between 50 and 500 potential causative genes. 
(This assumes 1 megabase mapping resolution and one gene per 20 kb). 
The polypeptides, their fragments or other derivatives, or analogs thereof, 
or cells expressing them can be used as an immunogen to produce antibodies 
thereto. These antibodies can be, for example, polyclonal or monoclonal 
antibodies. The present invention also includes chimeric, single chain, 
and humanized antibodies, as well as Fab fragments, or the product of an 
Fab expression library. Various procedures known in the art may be used 
for the production of such antibodies and fragments. 
Antibodies generated against the polypeptides corresponding to a sequence 
of the present invention can be obtained by direct injection of the 
polypeptides into an animal or by administering the polypeptides to an 
animal, preferably a nonhuman. The antibody so obtained will then bind the 
polypeptides itself. In this manner, even a sequence encoding only a 
fragment of the polypeptides can be used to generate antibodies binding 
the whole native polypeptides. Such antibodies can then be used to isolate 
the polypeptide from tissue expressing that polypeptide. 
For preparation of monoclonal antibodies, any technique which provides 
antibodies produced by continuous cell line cultures can be used. Examples 
include the hybridoma technique (Kohler and Milstein, 1975, Nature, 
256:495-497), the trioma technique, the human B-cell hybridoma technique 
(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma 
technique to produce human monoclonal antibodies (Cole, et al., 1985, in 
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). 
Techniques described for the production of single chain antibodies (U.S. 
Pat. No. 4,946,778) can be adapted to produce single chain antibodies to 
immunogenic polypeptide products of this invention. Also, transgenic mice 
may be used to express humanized antibodies to immunogenic polypeptide 
products of this invention. 
The present invention will be further described with reference to the 
following examples; however, it is to be understood that the present 
invention is not limited to such examples. All parts or amounts, unless 
otherwise specified, are by weight. 
In order to facilitate understanding of the following examples certain 
frequently occurring methods and/or terms will be described. 
"Plasmids" are designated by a lower case p preceded and/or followed by 
capital letters and/or numbers. The starting plasmids herein are either 
commercially available, publicly available on an unrestricted basis, or 
can be constructed from available plasmids in accord with published 
procedures. In addition, equivalent plasmids to those described are known 
in the art and will be apparent to the ordinarily skilled artisan. 
"Digestion" of DNA refers to catalytic cleavage of the DNA with a 
restriction enzyme that acts only at certain sequences in the DNA. The 
various restriction enzymes used herein are commercially available and 
their reaction conditions, cofactors and other requirements were used as 
would be known to the ordinarily skilled artisan. For analytical purposes, 
typically 1 .mu.g of plasmid or DNA fragment is used with about 2 units of 
enzyme in about 20 .mu.l of buffer solution. For the purpose of isolating 
DNA fragments for plasmid construction, typically 5 to 50 .mu.g of DNA are 
digested with 20 to 250 units of enzyme in a larger volume. Appropriate 
buffers and substrate amounts for particular restriction enzymes are 
specified by the manufacturer. Incubation times of about 1 hour at 
37.degree. C. are ordinarily used, but may vary in accordance with the 
supplier's instructions. After digestion the reaction is electrophoresed 
directly on a polyacrylamide gel to isolate the desired fragment. 
Size separation of the cleaved fragments is performed using 8 percent 
polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 
8:4057 (1980). 
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide 
or two complementary polydeoxynucleotide strands which may be chemically 
synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus 
will not ligate to another oligonucleotide without adding a phosphate with 
an ATP in the presence of a kinase. A synthetic oligonucleotide will 
ligate to a fragment that has not been dephosphorylated. 
"Ligation" refers to the process of forming phosphodiester bonds between 
two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 
146). Unless otherwise provided, ligation may be accomplished using known 
buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 
.mu.g of approximately equimolar amounts of the DNA fragments to be 
ligated. 
Unless otherwise stated, transformation was performed as described in the 
method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973). 
EXAMPLE 1 
Bacterial Expression and Purification of soluble CysE 
The DNA sequence encoding CysE, ATCC # 97156, is initially amplified using 
PCR oligonucleotide primers corresponding to the 5' sequences of the 
processed CysE protein (minus the signal peptide sequence) and the vector 
sequences 3' to the CysE gene. Additional nucleotides corresponding to 
CysE were added to the 5' and 3' sequences respectively. The 5' 
oligonucleotide primer has the sequence 5' CGCCCATGGCGGCCG CAGGAG 3' (SEQ 
ID NO:3) contains an NcoI restriction enzyme site followed by CysE coding 
sequence starting from the presumed terminal amino acid of the processed 
protein. The 3' sequence 5' CGCAAGCTTTCACATCTGCAAAAAGTTGGC 3' (SEQ ID 
NO:4) contains complementary sequences to a HindIII site and is followed 
by CysE coding sequence. The restriction enzyme sites correspond to the 
restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, 
Inc. Chatsworth, Calif., 91311). pQE-9 encodes antibiotic resistance 
(Amp.sup.r), a bacterial origin of replication (ori), an IPTG-regulatable 
promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and 
restriction enzyme sites. pQE-9 was then digested with NcoI and HindIII. 
The amplified sequences were ligated into pQE-9 and were inserted in frame 
with the sequence encoding for the histidine tag and the RBS. The ligation 
mixture was then used to transform E. coli strain M15/rep 4 (Qiagen, Inc.) 
by the procedure described in Sambrook, J. et al., Molecular Cloning: A 
Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains 
multiple copies of the plasmid pREP4, which expresses the laci repressor 
and also confers kanamycin resistance (Kan.sup.r). Transformants are 
identified by their ability to grow on LB plates and ampicillin/kanamycin 
resistant colonies were selected. Plasmid DNA was isolated and confirmed 
by restriction analysis. Clones containing the desired constructs were 
grown overnight (O/N) in liquid culture in LB media supplemented with both 
Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a 
large culture at a ratio of 1:100 to 1:250. The cells were grown to an 
optical density 600 (O.D. .sup.600) of between 0.4 and 0.6. IPTG 
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final 
concentration of 1 mM. IPTG induces by inactivating the lacI repressor, 
clearing the P/O leading to increased gene expression. Cells were grown an 
extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell 
pellet was solubilized in the chaotropic agent 6 Molar Guanidine HCl. 
After clarification, solubilized CysE was purified from this solution by 
chromatography on a Nickel-Chelate column under conditions that allow for 
tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. 
Chromatography 411:177-184 (1984)). CysE was eluted from the column in 6 
molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 
3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione 
(reduced) and 2 mmolar glutathione (oxidized). After incubation in this 
solution for 12 hours the protein was dialyzed to 10 mmolar sodium 
phosphate. 
EXAMPLE 2 
Cloning and expression of CysE using the baculovirus expression system 
The DNA sequence encoding the full length CysE protein, ATCC # 97156, was 
amplified using PCR oligonucleotide primers corresponding to the 5' and 3' 
sequences of the gene: 
The 5' primer has the sequence 5' CGCGGATCCGCCATCATGGCGC GTTCGAACCTC 3' 
(SEQ ID NO:5) and contains a BamHI restriction enzyme site (in bold) 
followed by an efficient signal for the initiation of translation in 
eukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) and 18 
nucleotides of the CysE gene (the initiation codon for translation "ATG" 
is underlined). 
The 3' primer has the sequence 5' CGCGGATCCTCACATCT GCAAAAAGTTGGCTT 3' (SEQ 
ID NO:6) and contains the cleavage site for the restriction endonuclease 
BamHI and nucleotides complementary to the 3' non-translated sequence of 
the CysE gene. The amplified sequences were isolated from a 1% agarose gel 
using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, 
Calif.). The fragment was then digested with the endonuclease BamHI and 
then purified again on a 1% agarose gel. This fragment is designated F2. 
The vector pA2 (modification of pVL941 vector, discussed below) is used for 
the expression of the CysE protein using the baculovirus expression system 
(for review see: Summers, M. D. and Smith, G. E. 1987, A manual of methods 
for baculovirus vectors and insect cell culture procedures, Texas 
Agricultural Experimental Station Bulletin No. 1555). This expression 
vector contains the strong polyhedrin promoter of the Autographa 
californica nuclear polyhedrosis virus (AcMNPV) followed by the 
recognition sites for the restriction endonuclease BamHI. The 
polyadenylation site of the simian virus (SV)40 is used for efficient 
polyadenylation. For an easy selection of recombinant virus the 
beta-galactosidase gene from E.coli is inserted in the same orientation as 
the polyhedrin promoter followed by the polyadenylation signal of the 
polyhedrin gene. The polyhedrin sequences are flanked at both sides by 
viral sequences for the cell-mediated homologous recombination of 
co-transfected wild-type viral DNA. Many other baculovirus vectors could 
be used in place of pA2, such as pRG1 pAc373, pVL941 and pAcIM1 (Luckow, 
V. A. and Summers, M. D., Virology, 170:31-39). 
The plasmid was digested with the restriction enzyme BamHI and then 
dephosphorylated using calf intestinal phosphatase by procedures known in 
the art. The DNA was then isolated from a 1% agarose gel using the 
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). 
This vector DNA is designated V2. 
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA 
ligase. E.coli HB101 cells were then transformed and bacteria identified 
that contained the plasmid (pBacCysE) with the CysE gene using the enzyme 
BamHI. The sequence of the cloned fragment was confirmed by DNA 
sequencing. 
5 .mu.g of the plasmid pBacCysE was co-transfected with 1.0 .mu.g of a 
commercially available linearized baculovirus ("BaculoGold.TM. baculovirus 
DNA", Pharmingen, San Diego, Calif.) using the lipofection method (Felgner 
et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)). 
1 .mu.g of BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid pBacCysE 
were mixed in a sterile well of a microtiter plate containing 50 .mu.l of 
serum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). 
Afterwards 10 .mu.l Lipofectin plus 90 .mu.l Grace's medium were added, 
mixed and incubated for 15 minutes at room temperature. Then the 
transfection mixture was added drop-wise to the Sf9 insect cells (ATCC CRL 
1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium 
without serum. The plate was rocked back and forth to mix the newly added 
solution. The plate was then incubated for 5 hours at 27.degree. C. After 
5 hours the transfection solution was removed from the plate and 1 ml of 
Grace's insect medium supplemented with 10% fetal calf serum was added. 
The plate was put back into an incubator and cultivation continued at 
27.degree. C. for four days. 
After four days the supernatant was collected and a plaque assay performed 
similar as described by Summers and Smith (supra). As a modification an 
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was 
used which allows an easy isolation of blue stained plaques. (A detailed 
description of a "plaque assay" can also be found in the user's guide for 
insect cell culture and baculovirology distributed by Life Technologies 
Inc., Gaithersburg, page 9-10). 
Four days after the serial dilution, the virus was added to the cells, blue 
stained plaques were picked with the tip of an Eppendorf pipette. The agar 
containing the recombinant viruses was then resuspended in an Eppendorf 
tube containing 200 .mu.l of Grace's medium. The agar was removed by a 
brief centrifugation and the supernatant containing the recombinant 
baculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Four days 
later the supernatants of these culture dishes were harvested and then 
stored at 4.degree. C. 
Sf9 cells were grown in Grace's medium supplemented with 10% 
heat-inactivated FBS. The cells were infected with the recombinant 
baculovirus V-CysE at a multiplicity of infection (MOI) of 2. Six hours 
later the medium was removed and replaced with SF900 II medium minus 
methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours 
later 5 .mu.Ci of .sup.35 S-methionine and 5 .mu.Ci .sup.35 S cysteine 
(Amersham) were added. The cells were further incubated for 16 hours 
before they were harvested by centrifugation and the labelled proteins 
visualized by SDS-PAGE and autoradiography. 
EXAMPLE 3 
Expression of Recombinant CysE in COS cells 
The expression of plasmid, CysE HA is derived from a vector pcDNAI/Amp 
(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin 
resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by 
a polylinker region, an SV40 intron and polyadenylation site. A DNA 
fragment encoding the entire CysE precursor and a HA tag fused in frame to 
its 3' end was cloned into the polylinker region of the vector, therefore, 
the recombinant protein expression is directed under the CMV promoter. The 
HA tag corresponds to an epitope derived from the influenza hemagglutinin 
protein as previously described (I. Wilson, H. Niman, R. Heighten, A 
Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The 
infusion of HA tag to the target protein allows easy detection of the 
recombinant protein with an antibody that recognizes the HA epitope. 
The plasmid construction strategy is described as follows: 
The DNA sequence encoding CysE, ATCC # 97156, was constructed by PCR on the 
original EST cloned using two primers: the 5' primer 5' 
GCGCGGATCCACCATGGCGCGTTCG 3' (SEQ ID NO:7) contains a BamHI site followed 
by 12 nucleotides of CysE coding sequence starting from the initiation 
codon; the 3' sequence 5' GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTACAT 
CTGCACAAA 3' (SEQ ID NO:8) contains complementary sequences to an XbaI 
site, translation stop codon, HA tag and nucleotides of the CysE coding 
sequence (not including the stop codon). Therefore, the PCR product 
contains a BamHI site, CysE coding sequence followed by HA tag fused in 
frame, a translation termination stop codon next to the HA tag, and an 
XbaI site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were 
digested with BamHI and XbaI restriction enzyme and ligated. The ligation 
mixture was transformed into E. coli strain SURE (available from 
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, 
Calif. 92037) approximately ten pieces are placed in each flask. The flask 
the transformed culture was plated on ampicillin media plates and 
resistant colonies were selected. Plasmid DNA was isolated from 
transformants and examined by restriction analysis for the presence of the 
correct fragment. For expression of the recombinant CysE, COS cells were 
transfected with the expression vector by DEAE-DEXTRAN method (J. 
Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, 
Cold Spring Laboratory Press, (1989)). The expression of the CysE HA 
protein was detected by radiolabelling and immunoprecipitation method (E. 
Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor 
Laboratory Press, (1988)). Cells were labelled for 8 hours with .sup.35 
S-cysteine two days post transfection. Culture media was then collected 
and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 
0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 
37:767 (1984)). Both cell lysate and culture media were precipitated with 
an HA specific monoclonal antibody. Proteins precipitated were analyzed on 
15% SDS-PAGE gels. 
EXAMPLE 4 
Expression via Gene Therapy 
Fibroblasts are obtained from a subject by skin biopsy. The resulting 
tissue is placed in tissue-culture medium and separated into small pieces. 
Small chunks of the tissue are placed on a wet surface of a tissue culture 
flask, is turned upside down, closed tight and left at room temperature 
over night. After 24 hours at room temperature, the flask is inverted and 
the chunks of tissue remain fixed to the bottom of the flask and fresh 
media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, 
is added. This is then incubated at 37.degree. C. for approximately one 
week. At this time, fresh media is added and subsequently changed every 
several days. After an additional two weeks in culture, a monolayer of 
fibroblasts emerge. The monolayer is trypsinized and scaled into larger 
flasks. 
pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by the long 
terminal repeats of the Moloney murine sarcoma virus, is digested with 
EcoRI and HindIII and subsequently treated with calf intestinal 
phosphatase. The linear vector is fractionated on agarose gel and 
purified, using glass beads. 
The cDNA encoding a polypeptide of the present invention is amplified using 
PCR primers which correspond to the 5' and 3' end sequences respectively. 
The 5' primer containing an EcoRI site and the 3' primer having contains a 
HindIII site. Equal quantities of the Moloney murine sarcoma virus linear 
backbone and the EcoRI and HindIII fragment are added together, in the 
presence of T4 DNA ligase. The resulting mixture is maintained under 
conditions appropriate for ligation of the two fragments. The ligation 
mixture is used to transform bacteria HB101, which are then plated onto 
agar-containing kanamycin for the purpose of confirming that the vector 
had the gene of interest properly inserted. 
The amphotropic pA317 or GP+am12 packaging cells are grown in tissue 
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) 
with 10% calf serum (CS), penicillin and streptomycin. The MSV vector 
containing the gene is then added to the media and the packaging cells are 
transduced with the vector. The packaging cells now produce infectious 
viral particles containing the gene (the packaging cells are now referred 
to as producer cells). 
Fresh media is added to the transduced producer cells, and subsequently, 
the media is harvested from a 10 cm plate of confluent producer cells. The 
spent media, containing the infectious viral particles, is filtered 
through a millipore filter to remove detached producer cells and this 
media is then used to infect fibroblast cells. Media is removed from a 
sub-confluent plate of fibroblasts and quickly replaced with the media 
from the producer cells. This media is removed and replaced with fresh 
media. If the titer of virus is high, then virtually all fibroblasts will 
be infected and no selection is required. If the titer is very low, then 
it is necessary to use a retroviral vector that has a selectable marker, 
such as neo or his. 
The engineered fibroblasts are then injected into the host, either alone or 
after having been grown to confluence on cytodex 3 microcarrier beads. The 
fibroblasts now produce the protein product. 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings and, therefore, within the scope of the 
appended claims, the invention may be practiced otherwise than as 
particularly described. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 8 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 581 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 16..462 
- - (ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- - #peptide 
(B) LOCATION: 100..462 
- - (ix) FEATURE: 
(A) NAME/KEY: sig.sub.-- - #peptide 
(B) LOCATION: 16..99 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - ACGGCACTGA CGGCC ATG GCG CGT TCG AAC CTC CCG - #CTG GCG CTG GGC CTG 
51 
Met - #Ala Arg Ser Asn Leu Pro Leu Ala Leu Gly L - #eu 
-28 - # -25 - # -20 
- - GCC CTG GTC GCA TTC TGC CTC CTG GCG CTG CC - #A CGC GAT GCC CGG GCC 
99 
Ala Leu Val Ala Phe Cys Leu Leu Ala Leu Pr - #o Arg Asp Ala Arg Ala 
-15 - # -10 - # -5 
- - CGG CCG CAG GAG CGC ATG GTC GGA GAA CTC CG - #G GAC CTG TCG CCC GAC 
147 
Arg Pro Gln Glu Arg Met Val Gly Glu Leu Ar - #g Asp Leu Ser Pro Asp 
1 5 - # 10 - # 15 
- - GAC CCG CAG GTG CAG AAG GCG GCG CAG GCG GC - #C GTG GCC AGC TAC AAC 
195 
Asp Pro Gln Val Gln Lys Ala Ala Gln Ala Al - #a Val Ala Ser Tyr Asn 
20 - # 25 - # 30 
- - ATG GGC AGC AAC AGC ATC TAC TAC TTC CGA GA - #C ACG CAC ATC ATC AAG 
243 
Met Gly Ser Asn Ser Ile Tyr Tyr Phe Arg As - #p Thr His Ile Ile Lys 
35 - # 40 - # 45 
- - GCG CAG AGC CAG CTG GTG GCC GGC ATC AAG TA - #C TTC CTG ACG ATG GAG 
291 
Ala Gln Ser Gln Leu Val Ala Gly Ile Lys Ty - #r Phe Leu Thr Met Glu 
50 - # 55 - # 60 
- - ATG GGG AGC ACA GAC TGC CGC AAG ACC AGG GT - #C ACT GGA GAC CAC GTC 
339 
Met Gly Ser Thr Asp Cys Arg Lys Thr Arg Va - #l Thr Gly Asp His Val 
65 - # 70 - # 75 - # 80 
- - GAC CTC ACC ACT TGC CCC CTG GCA GCA GGG GC - #G CAG CAG GAG AAG CTG 
387 
Asp Leu Thr Thr Cys Pro Leu Ala Ala Gly Al - #a Gln Gln Glu Lys Leu 
85 - # 90 - # 95 
- - CGC TGT GAC TTT GAG GTC CTT GTG GTT CCC TG - #G CAG AAC TCC TCT CAG 
435 
Arg Cys Asp Phe Glu Val Leu Val Val Pro Tr - #p Gln Asn Ser Ser Gln 
100 - # 105 - # 110 
- - CTC CTA AAG CAC AAC TGT GTG CAG ATG TGATAAGTC - #C CCGAGGGCGA 
482 
Leu Leu Lys His Asn Cys Val Gln Met 
115 - # 120 
- - AGGCCATTGG GTTTGGGGCC ATGGTGGAGG GCACTTCACG TCCGTGGGCC GT - 
#ATCTGTCA 542 
- - CAATAAATGG CCAGTGCTGC TTCTTGCAAA AAAAAAAAA - # 
- # 581 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 149 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Ala Arg Ser Asn Leu Pro Leu Ala Leu Gl - #y Leu Ala Leu Val Ala 
28 -25 - # -20 - # -15 
- - Phe Cys Leu Leu Ala Leu Pro Arg Asp Ala Ar - #g Ala Arg Pro Gln Glu 
-10 - # -5 - # 1 
- - Arg Met Val Gly Glu Leu Arg Asp Leu Ser Pr - #o Asp Asp Pro Gln Val 
5 - # 10 - # 15 - # 20 
- - Gln Lys Ala Ala Gln Ala Ala Val Ala Ser Ty - #r Asn Met Gly Ser Asn 
25 - # 30 - # 35 
- - Ser Ile Tyr Tyr Phe Arg Asp Thr His Ile Il - #e Lys Ala Gln Ser Gln 
40 - # 45 - # 50 
- - Leu Val Ala Gly Ile Lys Tyr Phe Leu Thr Me - #t Glu Met Gly Ser Thr 
55 - # 60 - # 65 
- - Asp Cys Arg Lys Thr Arg Val Thr Gly Asp Hi - #s Val Asp Leu Thr Thr 
70 - # 75 - # 80 
- - Cys Pro Leu Ala Ala Gly Ala Gln Gln Glu Ly - #s Leu Arg Cys Asp Phe 
85 - # 90 - # 95 - #100 
- - Glu Val Leu Val Val Pro Trp Gln Asn Ser Se - #r Gln Leu Leu Lys His 
105 - # 110 - # 115 
- - Asn Cys Val Gln Met 
120 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - CGCCCATGGC GGCCGCAGGA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - CGCAAGCTTT CACATCTGCA AAAAGTTGGC - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - CGCGGATCCG CCATCATGGC GCGTTCGAAC CTC - # - # 
33 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - CGCGGATCCT CACATCTGCA AAAAGTTGGC TT - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - GCGCGGATCC ACCATGGCGC GTTCG - # - # 
25 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 52 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (genomic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - GCGCTCTAGA TCAAGCGTAG TCTGGGACGT CGTATGGGTA CATCTGCACA AA - # 
52 
__________________________________________________________________________