Human somatomedin carrier protein subunits and process for producing them; recombinant DNA molecules, hosts, processes and human somatomedin carrier protein-like polypeptides

This invention relates to polypeptides that are human somatomedin carrier protein subunits and to processes for producing them. The carrier protein subunits bind to human somatomedin-like polypeptides also known as insulin-like growth factors. The process involves preparation from a human serum fraction, Cohn IV-1, by a molecule of various chromatographic steps. This invention also relates to DNA molecules encoding human somatomedin carrier protein-like polypeptides, recombinant DNA molecules, hosts, processes for producing carrier protein-like polypeptides, human somatomedin carrier protein-like polypeptides produced using those molecules, hosts and processes. The invention relates to DNA molecules and their expression in appropriate hosts. The recombinant DNA molecules contain DNA molecules that code for polypeptides which have a biological activity of the human carrier protein or a human carrier protein subunit capable of binding somatomedins. The DNA molecules, recombinant DNA molecules, hosts, and processes of this invention may be used in the production of polypeptides useful in a variety of therapeutic, diagnostic, and other useful applications.

FIELD OF THE INVENTION 
This invention relates to human somatomedin carrier protein subunits and to 
processes for producing them. More particularly, this invention relates to 
carrier protein subunits that bind to human somatomedin-like polypeptides, 
also known as insulin-like growth factors. In addition, this invention 
relates to essentially pure human somatomedin carrier protein subunits. 
This invention also relates to processes of preparing such carrier protein 
subunits from human plasma. The process involves preparation from a human 
serum fraction. Cohn IV-1. by a sequence of various chromatographic steps. 
The carrier protein subunits and methods of this invention may be used in 
a variety of therapeutic. diagnostic or other useful applications. 
This invention also relates to DNA molecules encoding human somatomedin 
carrier protein-like polypeptides, recombinant DNA molecules, hosts 
transformed with such molecules, processes for producing human somatomedin 
carrier protein-like polypeptides, and human somatomedin carrier 
protein-like polypeptides produced using those molecules, hosts and 
processes. More particularly, the invention relates to DNA molecules and 
their expression in appropriate hosts. The recombinant DNA molecules 
contain DNA molecules that code for polypeptides which have a biological 
activity of the human carrier protein. As will be appreciated from the 
disclosure to follow, the DNA molecules, recombinant DNA molecules, hosts, 
and processes of this invention may be used in the production of 
polypeptides useful in a variety of therapeutic, diagnostic, and other 
useful applications. 
BACKGROUND OF THE INVENTION 
Somatomedins (also sometimes referred to as "SMs") are hormones having 
useful biological properties. SMs are polypeptides having a molecular 
weight of approximately 7,500 daltons. SMs (a) mediate the 
growth-promoting effects of growth hormone (also sometimes referred to as 
"GH"). (b) have weak insulin-like activity (and for that reason are also 
called "insulin-like growth factors"or "IGFs"). (c) are mitogenic for a 
variety of skeletal and other tissues and (d) are transported in plasma 
bound to a large carrier protein. There are two SM compositions in humans. 
SM-C is a basic polypeptide and is sometimes referred to as SM-C. SM-C 
mediates the growth promoting actions of GH after birth. SM-A is a mixture 
primarily of a polypeptide known as IGF-II and variable amounts of a 
modified form of SM-C. Spencer, E.M., et al., "The Identity Of Human 
Insulin-like Growth Factors I and II With Somatomedins C and A With Rat SM 
I and II"in Insulin-like Growth Factors/Somatomedins; ed. Spencer, E.M. 
(Walter de Gruyter 1983). IGF-II is less GH dependent and may have a role 
in fetal growth. 
SMs may be useful in vivo to stimulate bone formation (for example, in 
treatment of osteoporosis). wound healing, and the growth of animals and 
GH-deficient humans. Serum levels of SM-C are measured to diagnose 
acromegaly, pituitary gigantism. GH deficiency, and other growth related 
conditions. Spencer, E.M., "Somatomedins"in Basic and Clinical 
Endocrinology, eds. Greenspan F. S. and Forsham, P. H. (1986), p. 89, 
Appleton-Century-Crofts. SMs are also employed to stimulate in vitro the 
proliferation of a variety of cells in tissue culture and, therefore, are 
useful in the study of the regulation of normal and abnormal cell growth. 
SMs produced by certain breast and kidney cancer cells may stimulate the 
proliferation of both the cancer cells and the vascular and fibrous 
tissues required to support the growth of the cancer tissues. Spencer, E. 
M. et al., "Possible Auto-stimulation of Human Mammary Carcinoma Growth by 
Somatomedins,"Annals of the N.Y. Acad. Sci., 464, p. 448 (1986): Huff. 
K.K., et al., "Secretion of Insulin-like Growth Factor-I-related Protein 
by Human Breast Cancer Cells."Cancer Research 46, pp. 4613-4619 (1986). 
Blocking the action of SMs may be useful to control the growth of these 
cancers. 
Human SMs appear to be transported and regulated in vivo by other proteins. 
Hintz, R. L. et al., "Demonstration of Specific Plasma Protein Binding 
Sites For Somatomedin,"J. Clin. Endocrinol. Metab. 45, p. 988 (1977). 
These proteins appear to bind to the SMs and regulate the biological 
activity of the SMs in vivo. Gel filtration of human serum at neutral pH 
has shown that 95% of the immunoreactive SM-C activity, and probably 
IGF-II activity, elutes at about 150,000 to 160,000 daltons (150-160 
kilodaltons or "kDa") with a minor amount in the range of 35-50 kDa. Only 
a very small amount of immunoreactive activity elutes at 7.5 kDa, where 
free SMs should appear Smith, G. L., Molecular and Cellular Endocrinology 
34, p. 83-89 (1984). This indicates that SMs are complexed with larger 
proteins in plasma. 
At least two different classes of proteins or protein complexes in human 
plasma have been reported to bind SMs. Drop. S. L. et al., "Immunoassay Of 
A Somatomedin-binding Protein From Human Amniotic Fluid: Levels In Fetal, 
Neonatal, And Adult Sera."J. Clin. Endocrinol. Metab. 59, p. 908 (1984): 
Wilkins, J. R. et al., "Affinity-labeled Plasma 
Somatomedin-C+/Insulin-like Growth Factor I Binding Proteins."J. Clin. 
Invest. 75 p. 1350 (1985). This description refers to one class of those 
native proteins or protein complexes as the SM "Carrier Protein"for its 
function appears to be the transport of SMs. This term is not intended to 
indicate that the carrier protein is a single protein. There may be more 
than one carrier protein and it may be a protein complex. This description 
refers to the other class as the "Amniotic Fluid Binding Protein"or 
"AFBP." There may be more than one AFBP. It is also possible that 
additional classes of proteins or protein complexes that bind SMs will be 
discovered. 
Carrier protein activity, like SM-C activity, is GH-dependent, being low in 
persons with GH deficiency and elevated in patients with GH-producing 
tumors, a condition known as acromegaly. White. R. M., et al., "The Growth 
Hormone Dependence Of Somatomedin-binding Protein In Human Serum," J. Clin 
Endocrinol Metab. 53, p. 49 (1981). The carrier protein displays 
biological properties indicative of potentially valuable uses. In vivo, 
when SMs bind to carrier protein, the half-life of the SMs is reported to 
increase from approximately one hour to up to about 24 hours depending on 
the animal species tested (Cohen. K. L. et al., "The Serum Half-life Of 
Somatomedin Activity: Evidence For Growth Hormone Dependence, " Acta 
Endocrinol. 83, p. 243 (1976)), and the SMs are rendered inactive until 
released. Studies in other model systems suggest that impure preparations 
containing the carrier protein (a) abolish the metabolic action of the SMs 
on the perfused rat heart (Meuli C., et al., "NSILA-carrier Protein 
Abolishes The Action Of Nonsuppressible Insulin-like Activity (NSILA-s) On 
Perfused Rat Heart," Diabetoloqia 14, p. 255 (1978)). (b) inhibit the 
mitogenic effect of the SMs on cells in culture (Knauer. D. J., Proc. 
Natl. Acad. Sci. U.S.A., 77, pp. 7252-7256 (1980) and Kuffer. A. D., et 
al., "Partial Purification Of A Specific Inhibitor Of The Insulin-like 
Growth Factors By Reversed Phase High-performance Liquid Chromatography." 
J. of Chromatography. 336, pp. 87-92 (1984) and (c) block the insulin-like 
activity of SMs on rat adipose tissue (Zapf. J., et al., "Inhibition Of 
The Action Of Nonsuppressible Insulin-like Activity On Isolated Rat Rat 
Cells By Binding To Its Carrier Protein," J. Clin Invest. 63, p. 1077 
(1979). Partially pure preparations of the carrier protein have been used 
with radiolabeled SMs in research to conduct competitive binding assays 
for measuring SMs. Moses, A. C., et al., Endocrinology 104, p. 536 (1979). 
Because of their valuable biological properties, there have been many 
efforts to isolate and characterize the carrier protein or the subunits of 
the carrier protein responsible for that activity. Prior to this 
invention, all attempts to isolate and characterize in pure form the 
carrier protein or its active subunits have failed. This is due in part to 
the low concentration of carrier protein in plasma. A successful 
purification procedure also had to solve the problems of loss of activity 
because of enzymatic digestion and instability of the carrier protein, 
especially to changes in pH. Purification of the carrier protein subunits 
is further complicated by the presence in plasma of the AFBP, which also 
binds to somatomedins. 
The carrier protein is a glycoprotein serum at nautral pH, it is bound with 
SMs and the complex has a molecular weight of about 150-160 kDa when 
measured by gel filtration. The molecular weight of the carrier protein 
complex at neutral pH has also been determined by other methods to be 
about 125 kDa. Gel filtration chromatography of serum or plasma under acid 
conditions has been reported to separate bound SMs from the carrier 
protein and to give rise to a unit of the carrier protein that has a 
molecular weight of about 40-50 kDa. That unit also binds to somatomedins. 
Hintz, R. L., et al., "Demonstration Of Specific Plasma Protein Binding 
Sites For Somatomedin," J. Clin. Endocrinol. Metab. 45, p. 988 (1977). 
Since the 40-50 kDa acid-stable unit cannot be induced to reform the 
150-160 kDa carrier protein complex, others have suggested that the 
carrier protein may also be composed in part of an acid-labile unit that 
does not itself bind to somatomedins. Moses, A. C., et al., Endocrinology 
104, p. 536 (1979). Furlanetto reported treating serum with a 35-55% 
ammonium sulfate solution, isolating the precipitate, dissolving the 
precipitate in 0.05M Tris. pH 8.20 and chromatographing on DEAE Sephadex 
A-50 with Tris buffers. Furlanetto. R. W., "The Somatomedin C Binding 
Protein: Evidence For A Heterologous Subunit Structure," J. Clin, 
Endocrinol Metab. 51, p. 12 (1980). Furlanetto did not disclose any 
further purification. Rather. Furlanetto conducted experiments with 
various fractions to confirm his view that the somatomedin-C binding 
activity in serum is composed of at least two units, one has a Stokes' 
radius of 36 .ANG. and binds SM-C (the so-called acid stable unit) and the 
other had Stokes' radius of 30-38 .ANG. and does not bind SM-C (the 
so-called acid labile unit)). 
Wilkins identified, by affinity labeling, plasma proteins that complexed 
with SM-C. Wilkins. J. R., et al., "Affinity-labeled Plasma Somatomedin-C/ 
Insulin-like Growth Factor I Binding Proteins." J. Clin. Invest., 75, p. 
1350 (1985). .sup.125 I-SM-C was covalently cross-linked to proteins that 
bound SM-C in whole plasma and in Sephadex G-200 fractions of plasma. 
Following sodium dodecylsulfate polyacrylamide gel electrophoresis and 
autoradiography, the AFBP was identified in addition to species of about 
160, 110, 80, 50 and 25 kDa, Wilkins et al. hypothesized that the 160 kDa 
carrier protein complex consisted of 6 approximately 25 kDa (24-28 kDa) 
subunit complexes, each composed of the subunit plus SM-C. However, 
Wilkins et al., did not report isolation or purification of this 25 kDa 
subunit. Another worker proposed, but did not establish, a slightly larger 
subunit structure. Daughaday. W. H., et al., "Characterization Of 
Somatomedin Binding in Human Serum By Ultracentrifugation And Gel 
Filtration," J. Clin. Endocrinol. Metab. 55, p. 916 (1982). 
Several workers have reported unsuccessful attempts to isolate the 
acid-stable 40-50 kDa carrier protein unit from human plasma. Draznin et 
al., reported a material containing only 1% SM binding activity and did 
not disclose whether this material originated from carrier protein or 
AFBP. Draznin, B., et al., in "Somatomedins and Growth," eds. G. Giordano 
et al. (Academic Press 1979) pp. 149-160. Fryklund et al., fractionated 
fresh frozen human plasma by polyethylene glycol precipitation, 
carboxymethyl-Sephadex chromatography, and gel filtration. Fryklund. L., 
et al., in Hormones and Cell Culture, eds G. H. Sato et al. (Cold Spring 
Harbor Laboratory 1979) pp. 49-59. Fryklund et al., proposed that the 
carrier protein consisted of 2 dissimilar chains of 35 and 45 kDa. 
Fryklund et al., disclosed that glycine was released by N-terminal 
molecule analysis, but did not identify from which chain it orginated or 
whether both ended in glycine. The reported binding activity of the 
Fryklund et al. preparation was very low and purity was not reported. 
Fryklund et al. did not establish whether the carrier protein or the AFBP 
was present in their preparation. Morris et al., reported obtaining crude 
SM binding fractions by acetic acid extraction of human Cohn fraction IV. 
incubation with .sup.125 I-IGF-I and chromatography on Sephacryl S-200. 
Morris, D. H., et al., "Structure of Somatomedin-binding Protein: Alkaline 
pH-Induced Dissociation of an Acid-Stable. 60,000 Molecular Weight Complex 
Into Smaller Components," Endocrinology 111, pp. 801-805 (1982). Morris et 
al. described fractions containing bound radioactive SM-C with apparent 
molecular weights of 60,000 and 46,000. Morris et al. reported that 
exposing these fractions to pH 8.0 resulted in a shift of .sup.125 I-IGF-I 
binding activity from 60,000 and 46,000 daltons to fractions with 
complexes of 46,000 and 30,000. These fractions were not further purified. 
Martin et al. reported preparing a polyclonal antibody to the acid-stable 
unit. The latter was isolated by extracting human Cohn fraction IV with 2M 
acetic acid. 75mM NaCl. After removal of SMs by adsorption to SP-Sephadex, 
the acid stable unit was obtained by IGF-II-Affinity Chromatography and 
used for immunization. Martin et al. disclosed that HPLC could further 
purify the acid stable unit. No data was supplied to establish the purity 
of their final product. Martin. J. L., et al. "Antibody Against 
Acid-Stable Insulin-Like Growth Factor Binding Protein Detects 150,000 
Molecular Weight Hormone-Dependent Complex In Human Plasma," J. Clin. 
Endocrinol. Metab. 261, pp. 799-801 (1985). Kuffer et al. reported a 
partial purification of what he described as an inhibitor of insulin-like 
growth factors (SMs). Kuffer. A. D. et al., "Partial Purification of A 
Specific Inhibitor of the Insulin-Like Growth Factors By Reverse Phase 
High-Performance Liquid Chromatography." J. of Chromatography, 336, pp. 
87-92 (1984). Kuffer et al. prepared SM inhibitors having a molecular 
weight of 16,000 to 18,000 from Cohn fraction IV-1 by ion exchange 
chromatography and sequential gel chromatography under acid conditions on 
Sephadex G-75 and Bio-Gel P-30 columns. After affinity chromatography and 
high performance liquid chromatography. Kuffer et al. obtained the 
"inhibitory activity" as two peaks of activity. corresponding "to a major, 
apparently homogeneous, protein peak and a minor heterologous peak." 
Kuffer et al. did not report isolation of the activity of either peak. 
None of the above studies disclose a class of human carrier protein 
subunits capable of binding somatomedin-like polypeptides. In addition, 
none of these studies disclose any subunits of the carrier protein capable 
of binding SMs and purified to homogeneity. Purity is required to 
establish that the carrier protein has been isolated instead of the AFBP 
or a contaminant and to study biologic activity. An impure preparation may 
contain enzymes, causing the product to be unstable, and easily degraded 
or denatured. Impure preparations also cannot be used in animals and 
humans, because many impurities present in original serum or produced as a 
result of the purification procedures, are antigenic and could produce 
unwanted biologic effects. For example, human use in osteoporosis requires 
removal of all contaminants, which may be antigenic or have adverse 
biologic effects. 
Other workers have isolated a different protein capable of binding SMs and 
obtained from mid-gestational amniotic fluid of humans, the amniotic fluid 
binding protein or "AFBP." The AFBP is not the carrier protein or a 
subunit of the carrier protein. Wilkins. J. R. et al., "Affinity-labeled 
Plasma Somatomedin-C/Insulin-like Growth Factor I Binding Proteins," J. 
Clin. Invest. 75. p. 1350 (1985). The AFBP (a) is smaller than the 
so-called acid-stable unit of the carrier protein, having a molecular 
weight in the range 32-40 kDa. (b) is not glycosylated, (c) differs from 
the carrier protein subunits of this invention in its reported N-terminal 
molecule (Povoa. G. et al., "Isolation And Characterization of A 
Somatomedin-binding Protein From Mid-term Human Amniotic Fluid," Eur. J. 
Biochem. 144, pp. 199-204 (1984)). and (d) has different immunologic 
properties. Drop, S. L. S. et al., "Immunoassay of A Somatomedin-Binding 
Protein From Human Amniotic Fluid: Levels In Fetal. Neonatal and Adult 
Sera. J. Clin. Endocrinol. Metab. 59, p. 908 (1984); Martin. J. L. et al. 
supra. J. Clin. Endocrinol. Metab. 61, pp. 799-801 (1985). Antisera to the 
AFBP do not cross-react with the 150 kDa carrier protein or its 
acid-stable unit. Drop et al. reported that the AFBP levels determined by 
radio-immunoassay (RIA) were found to decrease during infancy and 
childhood --the inverse of the carrier protein and also, unlike the 
carrier protein, to have a significant diurnal variation. Enberg also 
isolated the AFBP from adult human plasma by four chromatographic steps: 
CM-Affigel blue, hydroxylapatite, fast protein liquid chromatography gel 
permeation and high performance liquid chromatography ("HPLC") 
hydroxylapatite. Enberg. G., "Purification of A High Molecular Weight 
Somatomedin Binding Protein From Human Plasma," Biochem. and Biophy. Res. 
Commun., 135, pp. 178-82 (1986). Enberg reported a "possible" N-terminal 
molecule, Ala-Pro-Trp-. demonstrating that the AFBP was isolated, not the 
150 kDa carrier protein as Enberg erroneously concluded. 
Proteins that bind SMs have also been identified in cell culture extracts 
(e.g., Adams, S.O., et al. Endocrinology 115, pp. 520-526 (1984)). Thus 
far, the carrier protein has not been isolated. Spencer first showed that 
primary cultures of liver cells produced a protein that complexes with 
SMs. Spencer, E. M, "The Use Of Cultured Rat Hepatocytes To Study The 
Synthesis Of Somatomedin And Its Binding Protein." FEBS Letters. 99, p. 
157, (1979). Subsequently, several cell types, normal and abnormal, have 
been found to synthesize a protein that complexes with SMs. Cultured 
Buffalo rat liver tumor cells (BRL 3A) produce a 33 kDa SM binding protein 
that differs from the carrier protein by antibody reactivity. N-terminal 
amino acid molecule, and absence of glycosylation. Lyons R. M. et al., 
Characterization of Multiplication-Stimulatory Activity "MSA" Carrier 
Protein." Molecular and Cellular Endocrinol. 45, pp. 263-70 (1986). 
Mottola. C. et al., J. of Biol. Chem., 261, pp. 1180-88 (1986). Romanus et 
al. reported that antibodies to this binding protein cross-reacted with a 
protein present in fetal serum but not adult rat serum. Romanus, J. A. et 
al., "Insulin-like Growth Factor Carrier Proteins In Neonatal And Adult 
Rat Serum Are Immunologically Different: Demonstration Using A New 
Radioimmunoassay For The Carrier Protein From BRL-3A Rat Liver Cells," 
Endocrinology, 118, p. 1743 (1986). The BRL-3A binding protein may be the 
rodent equivalent of the AFBP. but the N-terminal molecule data show no 
similarity between the two molecules. 
Many proteins and polypeptides have been produced by use of recombinant DNA 
techniques. There is no published report of production of carrier 
protein-like polypeptides in this manner. There are numerous obstacles to 
using the techniques of recombinant DNA technology to clone and express a 
carrier protein-like polypeptide gene. Obtaining a gene encoding a carrier 
protein-like polypeptide is difficult for a variety of reasons. Prior to 
the invention, the protein sequences of the carrier protein and the 
carrier protein subunits were unknown and, therefore, DNA molecules that 
would code for the subunits were unknown. No human tissue source was 
established. Fibroblasts had been shown to produce small amounts of a 
large uncharacterized SM binding protein (Adams. S. O. et al. 
Endocrinology 115, pp. 520-526 (1984)). While liver is the major source of 
somatomedins, it had never been shown to produce the carrier protein. In 
addition, the liver is difficult to use to isolate mRNA, due to 
ribonucleases. The quantities of carrier protein in serum are very low. 
Thus, mRNA might be rare. The genome including a DNA molecule coding for 
the carrier protein may contain intervening sequences. For these and other 
reasons, many pitfalls faced the conventional approach to attempt to 
isolate a gene encoding a carrier protein-like polypeptide --namely, 
identifying a source of mRNA containing large amounts of the desired 
molecule, creating a library of cDNA from that mRNA, screening the library 
with oligonucleotide probes designed to hybridize with cDNA having the 
desired molecule, and isolating or assembling a gene from those cDNA 
molecules. 
DISCLOSURE OF THE INVENTION 
In this description, the following terms are employed: 
Somatomedin-like --A polypeptide displaying the biological activities of 
one of the human SMs or insulin-like growth factors, including but not 
limited to SM-C, SM-A, IGF-I and IGF-II. That polypeptide may have amino 
acids in addition to those of native human SMs or it may not include all 
the amino acids of native human SMs. 
Carrier Protein --A glycoprotein or complex of glycoproteins in human 
plasma, displaying the ability to regulate the biological activity of the 
human SMs in vivo by a process involving binding of the SM-like 
polypeptides, being growth hormone dependent, and exhibiting an apparent 
molecular weight of about 125,000-160,000 daltons in physiological pH 
conditions when complexed with SMs. The carrier protein may also be 
polymorphic. For example, cells of different individuals may produce 
carrier protein species which are physiologically similar, but 
structurally slightly different from the prototype. 
Subunit --A polypeptide fragment, part, or component of a larger protein 
unit. The term subunit is not confined to its customary meaning of a 
discrete polypeptide chain bound by covalent or any other types of bonds 
to another discrete polypeptide chain. 
Carrier Protein Subunits --A class of subunits of the carrier protein. 
Polypeptide --A linear chain of amino acids connected by peptide bonds. A 
polypeptide may also contain one or more disulfide bonds between cystines 
of the same amino acid chain. 
Carrier Protein-like Polypeptide --A polypeptide displaying a human 
somatomedin regulating biological activity of the carrier protein and 
being capable of binding somatomedin-like polypeptides. Preferably, a 
carrier protein-like polypeptide displays a somatomedin-C regulating 
activity of the carrier protein. A carrier protein-like polypeptide may be 
a carrier protein subunit capable of binding somatomedin-like 
polypeptides, if it possesses such somatomedin regulating activity. This 
polypeptide may include one or more amino acids in addition to those of 
the carrier protein or such carrier protein subunits. This polypeptide may 
not include all of the amino acids of the carrier protein or such carrier 
protein subunits because one or more amino acids have been deleted or 
because one or more amino acids have been substituted for others. Thus, a 
carrier protein-like polypeptide may have the amino acid sequence of the 
carrier protein or of a carrier protein subunit in which an amino acid 
residue has been added, deleted or substituted. A carrier protein-like 
polypeptide may have the natural glycosyltion of the carrier protein, may 
lack the natural glycosylation of the carrier protein, or may have 
glycosyltion different from the natural glycosylation of the carrier 
protein. Thus, a carrier protein-like polypeptide may be unaccompanied by 
the associated natural glycosylation of the carrier protein. This 
polypeptide preferably has a molecular weight of about 40,000-50,000 
daltons or less, if measured in a form accompanied by natural 
glycosylation. This polypeptide more preferably has a molecular weight of 
about 30,000 daltons or less, if measured in that form. 
Somatomedin-C ("SM-C" or "IGF-I") --The principle hormone regulating growth 
after birth. SM-C mediates the growth promoting action of GH and binds to 
the carrier protein. 
Nucleotide --A monomeric unit of DNA or RNA consisting of a sugar moiety 
(pentose), a phosphate, and a heterocyclic base. The four DNA bases are 
adenine ("A"), guanine ("G"), cytosine ("C"), and thymine ("T"). The four 
RNA bases are A, G, C, and uracil ("U"). 
DNA Molecule --A molecule other than the entire human genome composed of a 
sequence of nucleotides connected one to the other by phosphodiester bonds 
between the 3' and 5' carbons of adjacent pentoses. A DNA molecule may be 
composed of an isolated sequence of nucleotides that are part of the human 
genome. A DNA molecule may be composed of a single DNA molecule (commonly 
called "single stranded DNA") or two DNA molecules composed of 
complementary nucleotides (commonly called "double stranded DNA"). 
Recombinant DNA Molecule --A DNA molecule having at least one nucleotide 
sequence resulting from joining or adding together at least two DNA 
molecules. 
Genome --The entire DNA of a cell or a virus. It includes the genes coding 
for the polypeptides of the organism, as well as operators, promoters and 
ribosome binding and other interaction sites. 
Gene --A DNA molecule which encodes through its mRNA a sequence of amino 
acids of a specific polypeptide. 
cDNA --A double-stranded DNA molecule produced from an RMA molecule by 
using that RNA as a template for RNA-directed synthesis of the first DNA 
strand followed by using that DNA strand as a template for DNA-directed 
synthesis of the second DNA strand. 
Transcription --The process of producing mRNA from a gene. 
Translation --The process of producing a polypeptide from mRNA. 
Expression --The process of producing a polypeptide by transcription and 
translation. 
Plasmid --A nonchromosomal double-stranded DNA molecule comprising an 
intact "replicon" such that the molecule is replicated in a host organism. 
When the plasmid is placed within a single celled organism, the 
characteristics of that organism may be changed as a result of the DNA of 
the plasmid. A cell transformed by a plasmid is called a "transformant." 
Virus --DNA or RNA molecules in a protein envelope or coat capable of 
infecting a cell or organism. 
Phage or Bacteriophaoe --Bacterial virus. 
Vehicle or Vector --A plasmid, phage, mammalian virus, cosmid, or other DNA 
molecule which is able to be transformed into and to replicate in a host, 
having one or more sites at which such DNA molecules may be cut in a 
determinable fashion without loss of an essential biological function of 
the DNA, e.g., replication, production of coat proteins or loss of 
promoter or binding sites, and having a marker suitable for use in the 
identification of a transformed host, e.g., tetracycline resistance. 
Cloning --A process of obtaining a population of organisms, cells or DNA 
molecules derived from one such organism, cell or DNA molecule. 
Expression Control Secuence --A DNA sequence that controls and regulates 
expression of genes when operatively linked to those genes. They include 
the lac system, the trp system, the tac system, the trc system, major 
operator and promoter regions of phage .lambda., the T7 system, the 
control region of fd coat protein, the control sequences of SV-40, the 
actin system, the metallothionein system, the LTR (promoter-containing 
long terminal repeat of retroviruses) system, and other sequences known to 
control the expression of genes of prokaryotic or eukaryotic cells or 
organisms and their viruses or combinations thereof. 
Host, Host Organism or Host Cell --A prokaryotic or eukaryotic cell or 
organism capable of being transformed by a vehicle or vector. 
Carrier Protein Subunits 
The invention solves the problems referred to by making available human 
carrier protein subunits capable of binding somatomedin-like polypeptides. 
The ability of the carrier protein subunits of the invention to bind 
somatomedin-like polypeptides has been demonstrated by binding those 
subunits in vitro to somatomedin-C at about physiological pH. This binding 
activity demonstrates that the carrier protein subunits of the invention 
will bind somatomedin-like polypeptides in vivo, and provide substantially 
the transport and regulatory activity of the native carrier protein. When 
this description refers to the capability of the carrier protein subunits 
to bind somatomedin-like polypeptides, it is referring to this ability to 
bind such polypeptides in vitro or in vivo. The carrier protein subunits 
have no substantial binding activity for insulin. 
The carrier protein subunits of the invention each constitute a single 
polypeptide chain. The carrier protein subunits of the invention have an 
N-terminal amino acid molecule of the formula: 
##STR1## 
wherein R is cysteine or half-cystine. Half-cystine refers to an amino 
acid bound to another half-cystine amino acid in the same polypeptide 
chain by a disulfide bond. Because the carrier protein may be polymorphic, 
the amino acid molecule of the carrier protein subunits may also vary 
depending on the polymorphic character of the carrier protein. For 
example, the carrier protein subunits may contain a glycine ("Gly") 
residue in place of the alanine ("Ala") at position 5 from the N-terminal. 
Similarly, the Glu at position 14 from the N-terminal may sometimes be 
replaced in part by Phe. 
The carrier protein subunits of the invention have a range of molecular 
weights. The molecular weights of the carrier protein subunits referred to 
in this description are those determined by SDS-PAGE gel electrophoresis 
against proteins of known weight conducted in the presence of a suitable 
reducing agent such as .beta.-mercaptoethanol "BME." The known protein 
standards were 200,000 (myosin (H-chain)). 97,400 (phosphorylase b), 
66,200 (bovine serum albumin) 43,000 (ovalbumin). 25,700 
(.alpha.chymotrypsinogen), 18,400 (.beta.-lactoglobulin and 14,300 
(lysozyme). Carrier protein subunits having molecular weights of about 
15,000, 21,000, 26,000 and 30,000 daltons have been isolated and 
identified. The carrier protein subunits may differ in molecular weight 
because they were present in the carrier protein as polypeptides of that 
size or because of enzymatic digestion or break-down from other causes 
Whatever the source of these differences, the carrier protein subunits of 
the invention have a molecular weight of about 30,000 or less. The carrier 
protein subunits of the invention preferably have a molecular weight of 
about 15,000 to and including about 30,000 daltons. 
The carrier protein subunits of the invention are glycoproteins, as shown 
by their positive reaction to the periodic acid Schiff reagent and ability 
to bind concanavalin A cross-linked to agarose (Con-A Sepharose, 
Pharmacia). Binding to Con-A Sepharose is specific for glycoproteins 
containing glucose and mannose residues. Specific residues include 
.alpha.-D-mannopyranosyl and .alpha.-D-glucopyranosyl residues. Therefore, 
the carrier protein subunits are substantially glycosylated. 
The invention also provides essentially pure carrier protein subunits 
having SM binding activity. The carrier protein subunits of the invention 
are essentially free of other proteins, peptides, nucleotides, 
polysaccharides, lipids and salts. By virtue of the invention, it is 
possible to obtain those subunits in sufficient purity for use in human 
and animal therapeutic agents, as animal growth promotion agents, in human 
and other animal diagnostic reagents, and in human and other animal 
research applications. 
The invention also provides therapeutic compositions comprising an 
effective amount of at least one carrier protein subunit capable of 
binding somatomedin-like polypeptides, or pharmacologically acceptable 
salts thereof, and a pharamacologically acceptable carrier. The carrier 
protein subunit of such therapeutic compositions may be at least one 
essentially pure carrier protein subunit. Compositions of carrier protein 
subunits of the invention have many therapeutic uses involving the 
important biological properties of SMs. Compositions comprising the human 
carrier protein subunits may be useful in treatment of diseases involving 
increased, unregulated SM-dependent growth. Thus, the ability of the 
carrier protein subunits of the invention to inactivate SMs by binding 
permits a new therapy of several conditions. In such therapies, it is 
apparent that an effective amount of the carrier protein subunit is an 
amount sufficiently in excess of the biologically active, unregulated SMs 
to block or inactivate the SM activity. For example, an effective amount 
of carrier protein subunit may be 10 or more times the amount of 
biologically active SMs on a molar basis. For example, some cancers have 
been shown to produce SMs: fibrosarcomas, chondrosarcomas and hepatoma 
cell lines. De Larco, J. E., et al., "A Human Fibrosarcoma Cell Line 
Producing Multiplication Stimulating Activity "MSA"-related Peptides," 
Nature, 272, pp. 356-358 (1978). Breast and renal cancers produce a SM 
which autostimulates the growth of the cancer. Spencer, E. M. et al., 
"Possible Auto-stimulation Of Human Mammary Carcinoma Growth By 
Somatomedins," Annals New York Academy Sciences, 464, pp. 448-449 (1986). 
Since endothelial cell and fibroblast proliferation are also stimulated by 
SMs, SMs produced by breast cancers can act also as a paracrine and 
stimulate the growth of the supporting stromal tissue critical to tumor 
survival. Bar. R. S., et al., "Receptors For Multiplication-stimulating 
Activity on Human Arterial and Venous Endothelial Cells, J. Clin. 
Endocrinol. Metab. 52, p. 814, (1981 ): Clemmons. D. R., et al., "Hormonal 
Control Of Immunoreactive Somatomedin Production By Cultured Human 
Fibroblasts." J. Clin. Invest 67, p. 10 (1981). By blocking the action of 
SMs. administering the human carrier protein subunit of the invention 
would be expected to reduce the rate of tumor growth and additionally 
render the malignant cells more sensitive to other drugs. 
Carrier protein subunit therapy could also help prevent blindness secondary 
to diabetic proliferative retinopathy. Spencer and others have shown that 
SM-C seems to be one of the factors stimulating endothelial and fibroblast 
proliferation in diabetic retinopathy. Lorenzi, M., Spencer, E. M. et al., 
"Improved Diabetic Control. Growth Factors and Rapid Progression Of 
Retinopathy," New Enqland Journal of Medicine, 308, p. 160, (1983): 
Ashton, I. K., et al., "Plasma somatomedin in diabetics with retinopathy 
and joint contractures" in Insulin-Like Growth Factors/Somatomedins., ed. 
Spencer, E. M. (Walter de Gruyter). The ability of the carrier protein 
subunit to block this adverse effect of SM-C (and possibly also IGF-II) 
could be a useful new therapy. 
The carrier protein subunits of the invention are also useful to produce 
antibodies. The invention will enable pure carrier protein subunits to be 
used as the antigen to produce both polyclonal antibodies with high 
titers, high affinities or blocking properties, and monoclonal antibodies 
that are not now available. These antibodies could be used for 
immunoassays to make specific measurements, for blocking carrier protein 
activity, affinity chromatography and immunohistochemistry. 
The carrier protein subunits can also be used to develop the first 
procedure to measure the free level of SMs in body fluids. This method 
would improve current methods that can only measure total SMs because the 
free level is really what determines their biological activity. The 
carrier protein subunit antibody would be used to separate the SM-carrier 
protein complex from the free SMs in fluids. The free SMs could then be 
measured by, for example, RIA. 
This invention also provides a composition comprising at least one carrier 
protein subunit substantially complexed with at least one somatomedin-like 
polypeptide. Such a composition would have a variety of therapeutic 
applications. SMs possess biological activity which make them potentially 
useful in many therapeutic applications. However, to maintain the required 
steady level of SMs in plasma, multiple daily injections would have to be 
given because the half-life of SMs may be less than one hour in the free 
condition. This obstacle cannot be overcome by administering a larger 
dosage because (a) SMs are potent mitogens for subcutaneous, muscular, and 
vascular tissues (fibro-blasts, endothelial cells, muscle cells, 
adipocytes, and endothelial cells) and could produce local tissue 
proliferation. (b) large amounts of free SMs would cause hypoglycemia, and 
(c) the excessive amount of SMs required to maintain a steady plasma level 
would not be cost effective. 
SM could be delivered to target tissues in a safe, effective physiologic 
manner and their half-life significantly prolonged by complexing them to 
the carrier protein subunits of the invention. The SM in a SM-carrier 
protein subunit complex would not be mitogenic at injection sites or 
hypoglycemic. This complex could be formulated to provide controlled, 
long-term absorption. After transport to target tissues, dissociation 
would release SM. Thus, therapy would mimic the physiologic delivery 
system. Successful therapeutic and animal husbandry use of SM-C, IGF-II 
and other somatomedin-like polypeptides are permitted by a composition of 
at least one human somatomedin-like polypeptide and at least one carrier 
protein subunit. Compositions comprising one or more carrier protein 
subunit and one or more SMs would also be useful for treatment of diseases 
such as postmenopausal osteoporosis, other forms of osteoporosis, and 
human GH deficiency, as well as for healing wounds and increasing animal 
growth. Such composition would be used to deliver SM to bony tissues and 
stimulate the growth of bone. Dissociation of the SM from the carrier 
protein subunit-SM complex should stimulate osteoblasts to increase bone 
formation in postmenopausal osteoporosis, invade the porous matrix of a 
prosthetic joint thereby stabilizing the prosthesis, and to promote 
healing of un-united fractures. 
Therapeutic compositions comprising an effective amount of at least one 
carrier protein subunit capable of binding somatomodin-like polypeptides, 
or pharmacologically acceptable salts thereof, and a pharmacologically 
acceptable carrier and therapeutic processes using such compositions may 
also be useful in treating injuries or diseases in which the natural 
healing mechanism or response involves the presence of regulated levels of 
biologically active somatomedins. For example, such compositions may be 
useful in wound healing, where the natural physiological response involves 
the presence of endogenous SMs at the site of the wound. An effective 
amount of carrier protein subunit is an amount sufficient to prolong the 
half-life of the endogenous biologically active somatomedins. 
Compositions of at least one carrier protein subunit and SM-C can be used 
as an effective biodegradable growth-enhancer in anmal husbandry. 
Currently antibiotics and steroids are commercially important animal 
growth promoters. Because there are serious health concerns with both 
classes, new agents are being sought, especially biodegradable ones. GH 
has been investigated. However, the SM-C-carrier protein subunit complex 
may be much more effective, because SM-C is the direct mediator of the 
growth promoting effect of GH. SM-C is neither diabetogenic nor lipolytic. 
For the same reasons applied to postmenopausal osteoporosis, the SM-C 
would have to be administered in composition with the carrier protein 
subunit. 
For all of these reasons, there have been many attempts to determine the 
protein structure needed for carrier protein-like activity. None have 
identified and isolated the carrier protein subunits of this invention or 
isolated them in pure form. 
Another aspect of the invention is a process for producing the human 
carrier protein subunits from human plasma comprising (a) chromatographing 
the portions of Cohn fraction IV-1 that are soluble in an aqueous solution 
of pH of about 4.5 to 7.5 on a sulfopropyl derivative of a cross-linked 
dextran adsorbent by sequentially eluting with aqueous solutions of 
increasing pH; (b) chromatographing an acidic solution of pH less than 
about 4.0 of the fractions from step (a) that contain somatomedin binding 
activity on the same adsorbent as step (a) and collecting the pass-through 
fraction, or chromatographing the fractions from step (a) on a phenyl 
derivative of agarose by adsorption from a neutral solution of about 10% 
ammonium sulfate and eluting with about 0.5M sodium thiocyanate solution 
at about neutral pH; (c) chromatographing the fraction from step (b) 
containing somatomedin binding activity by gel filtration and eluting with 
an acidic aqueous solution; (d) chromatographing the fraction from step 
(c) containing somatomedin binding activity on a solid support 
cross-linked to substantially pure somatomedin-C by adsorbing at about 
neutral pH and eluting with an acidic aqueous solution: and (e) 
chromatographing the fraction from step (d) containing somatomedin binding 
activity by reverse phase high performance liquid chromatography. 
Recombinant DNA And Carrier in-Like Polypeptides 
The present invention also involves locating. identifying, and isolating 
DNA molecules that code for carrier protein-like polypeptides, recombinant 
DNA molecules, vectors, hosts and methods for the use of those molecules, 
vectors and hosts in the production of carrier protein-like polypeptides, 
that is, polypeptides displaying a somatomedin regulating activity of a 
carrier protein and being capable of binding somatomedin-like 
polypeptides. By virtue of this invention, it is possible to obtain 
carrier protein-like polypeptides for use in therapeutic and diagnostic 
compositions and methods. This invention allows the production of these 
polypeptides in amounts and by methods not available previously. This 
invention also involves producing these polypeptides essentially, and more 
preferably completely, free of other polypeptides naturally present in 
human plasma. 
As will be appreciated from the disclosure. the DNA molecules and 
recombinant DNA molecules of the invention contain genes that are capable 
of directing the expression, in an appropriate host, of carrier 
protein-like polypeptides. Replication of these DNA molecules and 
recombinant DNA molecules in appropriate hosts also permits the production 
in large quantities of genes coding for these polypeptides. The molecular 
structure and properties of these polypeptides and genes may thus be 
readily determined. The polypeptides and molecules are useful, either as 
produced in the host or after appropriate modification, in compositions 
and methods for improving the production of these products themselves and 
for use in therapeutic and diagnostic compositions and methods. 
A basic aspect of this invention is the provision of a DNA molecule 
comprising a gene which codes for a carrier protein-like polypeptide, 
namely one displaying a somatomedin regulating activity of the carrier 
protein and being capable of binding somatomedin-like polypeptides. Such a 
DNA molecule has been isolated in the sense that it is not the entire 
human genome. Such a DNA molecule is preferably free of introns. Such a 
DNA molecule is also preferably essentially free of genes which code for 
any other polypeptide coded for by the human genome. Preferably, such a 
gene codes for a polypeptide having a molecular weight of about 
40,000-50,000 daltons or less, if molecular weight is measured in a form 
accompanied by natural glycosylation. Such a gene may code for a 
polypeptide displaying a somatomedin regulating activity of the carrier 
protein, and more preferably, a somatomedin-C regulating activity of the 
carrier protein. Such a gene may also code for a carrier protein-like 
polypeptide that is a carrier protein subunit capable of binding 
somatomedin-like polypeptides, and more preferably a carrier protein 
subunit capable of binding somatomedin-C. 
The invention also provides a process for obtaining a DNA molecule, 
comprising preparing cDNA molecules from mRNA found in cells or tissues 
that produce the carrier protein, determining which of the cDNA molecules 
hybridize to one or more labelled polynucleotide probes based on the DNA 
sequence of FIG. 4, analyzing the cDNA molecules that hybridized, and 
obtaining a DNA molecule having a gene which codes for a carrier 
proteinlike polypeptide. In that process, a DNA molecule having the gene 
may be obtained by ligating one or more cDNA molecules that hybridized 
with other cDNA molecules. synthetic DNA molecules, or recombinant DNA 
molecules. The cDNA molecule which hybridizes to said probe may be a cDNA 
molecule selected from the group consisting of a human liver gene library, 
a human fibroblast gene library. a human placenta library, and a human 
epithelial library. In that process, the labelled polynucleotide probe may 
have the DNA sequence shown in FIG. 2a. The invention also includes a DNA 
molecule made by that process, and a DNA molecule which encodes a carrier 
protein-like polypeptide coded for by a DNA molecule obtainable by that 
process. 
The invention also provides an oligonucleotide probe having all or a 
portion of the DNA sequence of any one of the DNA molecules LCP, LCP 0.70, 
LCP 0.77, LCP 2.3, LCP 2.5, FCP 1.8 and FCP 2.5, which selectively 
hybridizes to a DNA molecule encoding a carrier proteinlike polypeptide. 
In addition, a DNA molecule of the invention may be selected from the group 
consisting of the DNA molecule LCP 0.70, LCP 0.77, LCP 2 3, LCP 2.5, FCP 
1.8 and FCP 2.5. DNA molecules which hybridize to any of the DNA molecules 
LCP 0.70, LCP 0.77, LCP 2.3, LCP 2.5, FCP 1.8 and FCP 2.5, and which code 
for a carrier proteinlike polypeptide, and DNA molecules which code for a 
polypeptide coded for by any of the foregoing DNA molecules. A preferred 
DNA molecule comprises a DNA molecule which is the carrier protein-related 
portion of LCP 2.3. Another recombinant DNA molecule comprises a DNA 
molecule which is the carrier protein-related portion of LCP 2.3, and DNA 
molecules which code for a polypeptide coded for by said portions of LCP 
2.3. 
Furthermore, a DNA molecule of the invention may comprise a gene which 
codes for a polypeptide having the sequence of amino acids -1 to 290 of 
FIG. 4. amino acids 1 to 290 of FIG. 4, or amino acids 27 to 290 of FIG. 
4. A DNA molecule may also comprise a gene which codes for a polypeptide 
having the sequence of amino acids 27 to 290 of FIG. 4 and having a 
methionine residue preceding amino acid 27. 
A DNA molecule may also comprise a gene which codes for a polypeptide 
having the sequence of amino acids 27 to 290 and having a sequence of 
amino acid residues preceding amino acid 27 that constitute a secretion, 
signal or other precursor sequence recognized by a host. 
These DNA molecules may be used to construct a recombinant DNA molecule in 
which such DNA molecules are operatively linked to an expression control 
sequence. Preferably, such a recombinant DNA molecule constitutes a vector 
or vehicle. The invention provides a method for producing a vector 
comprising introducing into a vector such a DNA molecule. That method may 
comprise the additional step of introducing into said vector an expression 
control sequence, so as to control and to regulate the expression of that 
DNA molecule. The expression control sequence may be a lac system, a trp 
system, a tac system, a trc system, a T7 system, major operator and 
promoter regions of phage .lambda., the control region of fd coat protein, 
the control sequences of SV-40, the actin system, the metallothionein 
system, the LTR (promoter containing long terminal repeat of retrovirus) 
system, and other sequences which control the expression of genes or 
prokaryotic or eukaryotic cells and their viruses and combinations 
thereof. 
The recombinant DNA molecules and vectors of this invention permit the 
production of carrier protein-like polypeptides in hosts. The invention 
also includes a host transformed with at least one of those recombinant 
DNA molecules or vectors. A transformed host may be strains of E. coli, 
Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus, other 
bacteria, yeast, fungi animal, insect or plant hosts and human tissue 
cells. 
The invention provides a method for producing a carrier protein-like 
polypeptide, comprising the steps of transforming an appropriate host with 
such a recombinant DNA molecule or vector, and culturing said host to make 
such a polypeptide. Preferably, the method includes the additional step of 
collecting said polypeptide. In this method, the host may be strains of E. 
coli, Pseudomonas, Bacillus subtilis, Bacillus stearothermophilus, other 
bacteria, yeasts, fungi, animal, insect or plant hosts, and human tissue 
cells. The method for producing such a polypeptide may also comprise the 
steps of culturing a host transformed by such a recombinant DNA molecule 
or vector. 
The invention also provides a polypeptide that is coded for on expression 
by a recombinant DNA molecule or vector described above. 
The invention also provides an essentially pure carrier protein-like 
polypeptide other than a carrier protein subunit capable of binding 
somatomedin-like polypeptides. Such an essentially pure polypeptide is 
preferably essentially free of substances naturally present in human 
serum. Such a polypeptide may be a mature carrier protein-like 
polypeptide. Such a mature polypeptide is one in which the amino acid 
residues constituting a secretion, signal or other precursor sequence are 
deleted. 
The invention provides an essentially pure polypeptide having the sequence 
of amino acids -1 to 290 of FIG. 4. 
It also provides an essentially pure polypeptide having the sequence of 
amino acids 1 to 290 of FIG. 4. The invention includes a polypeptide 
having the sequence of amino acids 27 to 290 and having a methionine 
residue preceding amino acid 27. It further provides an essentially pure 
polypeptide having the sequence of amino acids 27 to 290 of FIG. 4. 
The invention includes polypeptides having the sequence 27 to 290 in which 
one or more amino acid residues may have been added, deleted or 
substituted, so long as the polypeptide remains a carrier protein-like 
polypeptide. 
The invention includes a polypeptide having the sequence of amino acids -1 
to 290 of FIG. 4, and polypeptides that have a portion of that sequence 
and have a somatomedin regulating activity of the carrier protein and are 
capable of binding somatomedin-like polypeptides. 
The invention also provides a carrier protein-like polypeptide lacking the 
natural glycosylation of the carrier protein. 
The invention is also a therapeutic composition for inhibiting the effect 
of somatomedin-C in acromegaly, for inhibiting the growth of retinal blood 
vessels and fibrous tissues in diabetic retinopathy, for inhibiting growth 
of tall children, for inhibiting the growth of keloid scars, for 
inhibiting the growth of tissue in the orbit of the eyes in malignant 
exophthalmos or for stimulating the healing of human or animal wounds, 
comprising an effective amount of at least one such carrier protein-like 
polypeptide described above, or a pharmacologically-acceptable salt 
thereof, and a pharmacologically-acceptable carrier. The invention 
includes a method for inhibiting the growth of somatomedin-dependent 
cancers, for inhibiting the effect of somatomedin-C in acromegaly, for 
inhibiting the growth of retinal blood vessels and fibrous tissues in 
diabetic retinopathy, for inhibiting growth of tall children, for 
inhibiting the growth of keloid scars, for inhibiting the growth of tissue 
in the orbit of the eyes in malignant exophthalmos or for stimulating the 
healing of human or animal wounds, comprising administering an effective 
amount of such a composition. 
The invention is also embodied in a composition having at least one such 
carrier protein-like polypeptide described above substantially complexed 
with at least one somatomedin-like polypeptide. Such compositions may be 
used in a therapeutic composition for treating osteoporosis in humans, for 
stimulating the growth of bone, for stimulating animal growth, for 
stimulating the healing of human and animal wounds, or for stimulating the 
growth of patients with growth hormone deficiency, comprising an effective 
amount of such a composition. Such compositions may also be used in a 
method for treating such conditions comprising administering an effective 
amount of such a composition. 
The invention provides a recombinant DNA molecule having a DNA molecule 
including a gene which codes for such a carrier protein-like polypeptide 
linked to an expression control sequence and having a DNA molecule 
including a gene which codes for a somatomedin-like polypeptide 
operatively linked to an expression control sequence. A host may be 
transformed with at least one such recombinant DNA molecule to permit it 
to produce both types of polypeptides. 
A single vector may also be constructed to contain a DNA molecule which 
codes for at least one carrier protein-like polypeptide described above 
and a DNA molecule which codes for a somatomedin-like polypeptide each 
operatively linked to an expression control sequence. A host may be 
transformed with such a vector. A method for producing a composition 
comprising a complex of a carrier protein-like polypeptide and a 
somatomedinlike polypeptide involves transforming an appropriate host with 
such a vector and culturing said host to make said polypeptides. That 
method could include the additional step of collecting the polypeptides. 
That method could comprise simply culturing a host transformed with such a 
vector. A method for producing such a composition also involves 
transforming an appropriate host with at least one recombinant DNA 
molecule or vector having a DNA molecule which codes for a carrier 
protein-like polypeptide as described above, co-transforming such host 
with at least one recombinant DNA molecule or vector having a DNA molecule 
which codes for a somatomedin-like polypeptide, and culturing such host to 
produce both types of polypeptides. The invention also encompasses hosts 
transformed with at least one of each such type of recombinant DNA 
molecule or vector. 
Finally, monoclonal and polyclonal antibodies against such polypeptides may 
be produced. The polypeptides of the invention could also be used in a 
method for measuring the level of free somatomedins in human fluids 
comprising separating somatomedins complexed with such polypeptides from 
unbound somatomedins.

Assay for Somatomedin Binding Activity 
The somatomedin binding activity is measured by a protein binding assay 
employing a radiolabeled .sup.125 I-SM (SM-C or IGF-II) as the ligand. The 
amount of .sup.125 I-SM bound is compared to that of a standard 
preparation. 
The standard was prepared by gel filtration of a pool of human serum from 
10 normal donors. The serum. 35 ml, was added to 35 ml of 4 N acetic acid. 
After clarification, the sample was chromatographed on Sephadex G-50 
(5.times.100 cm) (fractionation range 1500 to 30,000) equilibrated with 1 
M acetic acid at a flow rate of 80 ml/hr. All fractions were assayed for 
somatomedin binding activity using .sup.125 I-SM-C. The binding activity 
appeared from K.sub.d O- 0.4. These fractions were lyophilized, 
redissolved in 1M acetic acid and rechromatographed to remove all traces 
of bound SMs. The final powder was redissolved in 35 ml of 0.1M phosphate 
buffer pH 7.0. aliquoted in 100 ul amounts, and stored at -26.degree. C. 
For each binding assay, a tube of this material was used as a reference 
that has arbitrarily been assigned a value of 1.0 U/ml. 
The assay method was that described by Zapf et al., ("Serum Levels of the 
Insulin-like Growth Factor (SM) and its Carrier," Acta Endocrinol. 95, p. 
505-517, (1980)). For samples where the carrier protein subunit was still 
complexed to SMs, the two were separated by Sephadex G-50 chromatography 
(0.9.times.110 cm) in 1M acetic acid. The binding activity peak (K.sub.d.1 
- 0.4) was then lyophilized, reconstituted in assay buffer and tested. For 
samples that did not contain bound SMs, the samples were either dialyzed 
against assay buffer and tested directly or, if the concentration of 
binding activity was low, dialyzed vs 0.1 M acetic acid, lyophilized and 
dissolved in a smaller volume of assay buffer. The assay buffer was 0.1M 
sodium phosphate pH 7.0 containing 0.2% human or bovine serum albumin 
which had been previously tested to ensure absence of competing activity. 
SM-C or IGF-II were iodinated by the method of Spencer. Grecu, E. O., E. 
M. Spencer. et al., "Serum Somatomedin Response to Human Growth Hormone 
Infusion in Patients with Diabetes Mellitus: Correlation with the Degree 
of Control of Diabetes." Am. J. Med. Sci., 287, pp. 7-10 (1984). Serial 
dilutions (2-or 4-fold) of samples and standard were assayed in 
triplicate. Assay tubes consisted of 100 ul of .sup.125 I-SM. 20,000 cpm. 
and 200 ul of the sample. The assay was carried out at 4.degree. C. for 16 
h although satisfactory results could be obtained with a 2 h incubation at 
room temperature. The bound .sup.125 I-SM was separated from the free by 
charcoal extraction. An ice cold solution, 0.8 ml, of 2% activated 
charcoal with 1% human (or bovine) albumin in 0.1 M phosphate buffer pH 
7.0 was added and the tubes vortexed for 15 minutes at 4.degree. C. After 
centrifugation, the supernatant was counted. The cpm bound were plotted 
against the log of the dose and the potency of the unknown related to that 
of the standard assigned a value of 1.0 U/ml. The specificity of binding 
was determined by incubating the sample with a large excess of an 
unlabeled SM. 
Other Somatomedin Binding Assays 
Dot-blot and Western assays may also be used to determine the existence of 
polypeptides with somatomedin binding activity. 
Dot Blot "Binding In Wells" Format 
The nitrocellulose membrane and 3-MM filter paper are first placed in water 
and subsequently soaked in PBS (10 mM NaPO.sub.4, pH 7.2. 0.15 M NaCl) for 
20-30 minutes. The filter paper and membrane are placed on the dot-blot 
apparatus, with the membrane on top of the filter paper. The apparatus is 
assembled and clamped according to manufacturer's instructions (Bio-Rad). 
The dot-blot apparatus contains 96 wells which makes it very convenient to 
process many samples simultaneously. Wells are rinsed with 200 ul PBS. 
Carrier protein-like polypeptides are diluted in PBS to the appropriate 
concentrations to make total volumes of 50 ul/well. Control and blank 
wells contain BSA (bovine serum albumin) or no protein, respectively. 
Samples are applied to wells and are allowed to flow through the membrane 
by gravity. Binding of the protein to the membrane is completed within 
30-60 minutes. The membrane is blocked with 200 ul/well 1% BSA in PBS. 
which is allowed to flow by gravity for 30 minutes, then is "pulled" by a 
vacuum through the membrane. Wells are washed three times with 100 ul TBS 
(50 mM Tris-HCl, pH 7.5, 0.15M NaCl). 0.1% Tween 20. .sup.125 I-SM-C 
(20,000-200,000 cpm) is added in 50 ul PBS per well. The apparatus is 
tightly covered with Parafilm and left at 4' for 1.5-2 hours. This step 
constitutes the binding of SM-C to carrier protein-like polypeptides. The 
apparatus is disassembled and the membrane washed in large volumes of TBS: 
TBS. 0.1% Tween 20; and TBS: each wash is 15 minutes at 4.degree. C. with 
gentle shaking. The membrane is air dried and exposed to Kodak X-Omat AR 
film with intensifying screens at -70.degree. C. for 1-6 hours. 
Dot Blot "Binding in Bag" Format 
Pretreatment of membrane, dot-blot apparatus assembly, and binding of 
protein to membrane is carried out as described above. Following binding 
of protein to membrane, the dot-blot apparatus is disassembled, and the 
membrane is air dried. The membrane is placed in a dish and washed at 4' 
with gentle shaking in the following solutions: TBS plus 3% NP40, for 30 
minutes; TBS plus 1% BSA. for 1 hour; TBS plus 0.1% Tween 20, for 10 
minutes. The membrane is placed in a bag with 6-10 ml binding solution 
(TBS. 1% BSA. 0.1% Tween 20). .sup.125 I-SM-C (2-20 million cpm) is added 
and binding proceeds at 4.degree. C. for 2 hours or overnight, with gentle 
shaking. This step constitutes the binding of SM-C to carrier protein-like 
polypeptides. The membrane is washed two times in large volumes of TBS. 
0.1% Tween 20 and two times in TBS alone. Each wash is done for 15 minutes 
at 4.degree. C. with gentle shaking. The membrane is air dried and exposed 
to Kodak X-Omat AR film with intensifying screens at -70.degree. C. for 
5-16 hours. 
Western 
Protein samples containing carrier protein-like polypeptides are loaded and 
run on polyacrylamide-SDS gels. Normally 12% gels are run which will allow 
for good separation of proteins between 10,000 and 70,000 daltons. 
Separation is accomplished by electrophoresis. Proteins within the gel are 
then blotted onto a nitrocellulose membrane, and the resultant membrane is 
air dried 5 minutes at 37.degree. C. The membrane, containing the bound 
proteins, is rinsed with TBS plus 3% NP40 at 4.degree. C. for 30 minutes. 
The membrane s nonspecific sites are blocked with 1% BSA in TBS at 
4.degree. C. for 2 hours. The membrane is rinsed with TBS plus 0.1% Tween 
20 at 4.degree. C. for 10 minutes. The membrane is probed with .sup.125 
I-SM-C by placing the membrane in a bag with 6-10 ml TBS. 1% BSA, 0.1% 
Tween 20 plus 500,000 cpm .sup.125 I-SM-C. The membrane is gently shaken 
overnight at 4.degree. C. to allow for binding between SM-C and carrier 
protein-like polypeptides immobilized on the membrane. The membrane is 
subjected to the following washes at 4.degree. C.: TBS plus 0.1% Tween 20, 
twice, for 15 minutes each: TBS. three times, for 15 minutes each. The 
membrane is air dried and exposed to Kodak X-Omat AR film with 
intensifying screens at -70.degree. C. for 5-16 hours. 
Process For Producing Carrier Protein Subunits From Plasma 
The procedure for producing the carrier protein subunits began with Cohn 
fraction IV-1. This is a human plasma fraction that contains about 10% of 
the plasma proteins and 40% of the original plasma carrier protein 
activity. It is a green-yellow paste, approximately 35% solids, much of 
which are denatured insoluble proteins and glycoproteins. Each kilogram of 
this paste contains approximately 10 mg of carrier protein. 
All assay buffers described below contained the following enzyme 
inhibitors, unless otherwise noted: 1 millimolar ("mM") 
phenylmethylsulfonyl fluoride ("PMSF"), 1 mM N-ethylmaleimide ("NEM"), and 
1 mM ethylenediaminetetraacetic acid ("EDTA"). Enzyme inhibitors were 
essential because either the carrier protein has inherent protease 
activity or at least one other plasma protease was co-purified through the 
affinity chromatography step. 
EXAMPLE 1 
(a) Ion Exchange Chromatography 
Fraction IV-1 was handled in 1 kg batches. One kg of fraction IV-1 was 
added to 10 liters of 40 mM ammonium acetate-acetic acid solution pH 5.65 
containing enzyme inhibitors and stirred overnight at 4.degree. C. The 
suspension was centrifuged and the supernatant was concentrated to about 1 
liter by ultrafiltration with a 10,000 MW semipermeable membrane. 
The entire concentrate was applied to a 10.times.25 cm column at 4.degree. 
C. of a sulfopropyl derivative of crosslinked dextran (SP-Sephadex, 
Pharmacia) previously equilibrated with 40 mM ammonium acetate-acetic acid 
buffer at pH 5.65. The column was washed with 5 liters of the same buffer, 
followed by 10 liters of 50 mM ammonium acetate pH 6.8. and finally 2 
liters of 50 mM ammonium acetate-ammonia at pH 9.6. The pH 9.6 eluate was 
collected and lyophilized. The recovery of SM binding activity in the 
lyophilized material determined by the binding assay was 20%. This 
constituted about a 10 fold purification. 
(b) Hydrophobic Interactio:1 Chromatography 
The lyophilized product with SM binding activity was dissolved in a buffer 
containing 10% ammonium sulfate and 50 mM tris-(hydroxymethyl) 
aminomethane ("Tris")-hydrochloride ("Tris-HCl") pH 7.5, dialyzed against 
the same buffer, and applied to a phenyl agarose column 
(Phenyl-Sepharose,Pharmacia). The column was eluted first with 1 liter of 
the same buffer, then with 2 liters of 50 mM Tris-HCl pH 7.5 containing 
0.5 M sodium thiocyanate ("NaSCN") and finally with 2 liters of 50 mM 
Tris, pH 9.0. The eluted fractions were collected and tested for UV 
absorption at 280 nM and for SM-binding activity in the binding assay. The 
SM binding activity appeared in the NaSCN fractions. These were 
lyophilized and then dialyzed against distilled water. A significant 
amount of precipitate appeared which was separated from the supernatant. 
This step resulted in a 20-fold purification with 70% recovery. 
(c) Gel Filtration 
The supernatant was lyophilized, dissolved in 0.5 M acetic acid and 
chromatographed on a 2.times.100 column of a cross-linked dextran gel 
(Sephadex G-150, Pharmacia) having a fractionation range of 5,000-230,000. 
Fractions containing SM binding activity were collected. The recovery of 
SM binding activity was 80-90% by binding assay and the fold purification 
was 5. 
(d) Affinity Chromatoqraphy 
A SM-C affinity column was first made by coupling SM-C previously purified 
from human plasma (Spencer et al., in Insulin-Like Growth 
Factors/Somatomedins, ed. Spencer. E. M., Walter de Gruyter 1983), p. 81) 
to a hydroxysuccinimidyl derivative of agarose (Affi-Gel 15, BioRad) at pH 
8.0 and 25.degree. for 2 hours. The combined carrier protein fractions 
from the previous step were dialyzed against 0.1 M sodium phosphate pH 
7.0, then applied to the SM-C affinity column. After a 15 ml wash with the 
same buffer, the SM binding activity was eluted with 10 ml of 0.5M acetic 
acid and lyophilized. 
The SM binding activity was next chromatographed on a cross-linked dextran 
gel (Sephadex G-100, Pharmacia) having a fractionation range from 
4,000-90,000 and equilibrated with 0.5M acetic acid. The fractions 
containing activity, as shown by the SM binding assay, were lyophilized. 
(e) High Performance Liquid Chromatography("HPLC") 
The lyophilized material was chromatographed by HPLC on a butylsilane 
(Vydac C.sub.4 RP (reverse phase)) column. The SM binding activity was 
eluted by a 0-60% linear gradient of acetonitrile in 0.1% trifluroacetic 
acid ("TFA"). A sharp peak of SM-C binding activity occurred at 39% 
acetonitrile and was collected. The SM binding activity in this peak 
appeared as a single band on 12.5% sodium dodecylsulfate-polyacrylamide 
gel electrophoresis ("SDS-PAGE") upon staining with a silver stain 
(BioRad). 
The carrier protein subunit isolated had a molecular weight of 
approximately 26 kDa as shown by SDS-PAGE in the presence of 
.beta.-mercaptoethanol. The overall yield of the carrier protein subunit 
was 4% of the original binding activity. 
The N-terminal amino acid molecule of this carrier protein subunit was 
determined by the method of Hunkapillar and Hood (Methods in Enzymology, 
91, p. 486, (1983)), using an automated gas phase sequenator (Beckman 
6300) to be: 
##STR2## 
with R indicating cysteine or half-cystine. This carrier protein subunit 
bound .sup.125 I-SM-C and was shown to be glycosylated by periodic acid 
Schiff ("PAS") staining. 
EXAMPLE 2 
(a) Ion Exchange Chromatography 
One kg of Cohn fraction IV-1 was extracted with 4 liters of 40 mM ammonium 
acetate-acetic acid buffer pH 5.65 with inhibitors (1 mM EDTA. 1 mM NEM, 
0.1 mM PMSF and 1 mg/1 aprotinin) overnight at 4.degree. C. The protein 
solution was spun at 9,000.times.g for 30 minutes to separate precipitate 
from supernatant. The precipitate was reextracted with 4 liters of the 
above buffer for 4 hours. Supernatants from both extractions were 
combined. 
The supernatants were applied to a SP-Sephadex column (2000 ml resin) which 
had been equilibrated with the above buffer at 4.degree. C. After 
application, the column was washed with the same buffer until the 
A.sub.280 dropped below 1.0. The column was further washed with 50 mM 
ammonium acetate buffer, pH 6.8 with inhibitors until the A.sub.280 was 
below 1.0. Then the SM binding activity was eluted with 60 mM ammonium 
acetate-ammonia buffer, pH 9.6 with inhibitors. Finally, the column was 
cleaned with 60 mM ammonium acetate-ammonia, pH 9.6 with 1.0M NaCl. 
The extract from 1 kg Cohn fraction IV-1 gave about 5,000 units of 
SM-binding activity. In the pH 9.6 fractions about 7.5% of the activity 
was recovered, as determined by the binding assay. The weight of the 
fraction was approximately 5.5 g. 
(b) Ion Exchange Chromatography 
The pH 9.6 fraction from the previous column was dissolved in 130 ml of a 
1M acetic acid solution containing inhibitors (0.1 mM EDTA, PMSF, NEM and 
1 mg/1 aprotinin). The solution was dialyzed at 4.degree. C. overnight 
against the same buffer solution and applied to a 5.times.40 cm 
SP-Sephadex column, which had been previously equilibrated with the same 
buffer. The column was washed until A.sub.280 was approximately 0.2, then 
eluted with 60 mM ammonium acetate-ammonia, pH 9.6. with inhibitors. The 
SM binding activity was in the pass-through fraction which was dialyzed at 
4.degree. C. against distilled water overnight to precipitate some 
denatured proteins. After dialysis, the precipitate was removed by 
centrifugation at 9,000.times.g for 30 minutes and the supernatant 
freezed-dried. SM binding activity was recovered quantitatively in the 
soluble pass-through fraction, while SM-C was recovered in the pH 9.6 
fraction. 
(c) Gel Filtration 
An aliquot of the fraction (0.33 g) containing SM binding activity was then 
dissolved in a minimal amount of 0.5M acetic acid solution and applied to 
a 2.5.times.100 cm Sephadex G-100 column, which had been equilibrated 
under the same conditions. The column was eluted with 0.5M acetic acid. 
The A.sub.280 and SM binding activities of 5 ml fractions were measured. 
Those fractions exhibiting activity were pooled together and lyophilized. 
The purification was at this step five fold and the SM binding activity 
was recovered quantitatively. Several runs were required to process all 
the material. 
(d) Affinity Chromatography 
Eighty mg of fractions containing binding activity from the previous step 
were dissolved in 40 ml of 0.1M phosphate buffer, pH 7.0. with inhibitors 
and dialyzed against the same buffer for about 4 hrs. After dialysis, the 
solution was mixed with 3 ml SM-C-affinity column resin. The mixture was 
agitated gently at 4.degree. C. overnight to increase the binding. The 
resin was separated from the protein solution by passage through a column. 
The column was first washed with 50 ml of the phosphate buffer then eluted 
with 0.5M acetic acid. The SM binding activity (about 10 units) was dried 
in a vacuum centrifuge (Speed-Vac Concentrator. Savant Instruments). 
(e) HPLC 
The 10 units of recovered SM binding activity were dissolved in 1 ml 0.1% 
TFA solution. After injecting the sample onto a Vydak C4 RP column, the 
column was eluted with a 0-60% acetonitrile gradient in 60 minutes. The 
carrier protein peak appeared at approximately 39% acetonitrile, which was 
collected and lyophilized. The SM binding activity was recovered 
quantitatively and was approximately 60 micrograms. 
The SM binding activity appeared after silver staining as a single band on 
SDS-PAGE, with a molecular weight of about 15 kDa. The overall yield of 
this example was approximately 3%. 
The specific activity of the pure carrier protein subunit was determined to 
be 4 ug/unit where 1 unit is the amount in 1 ml of a standard plasma 
prepared from a pool of 10 normal men and women, as described above. 
For N-terminal molecule determination, the SM binding activity was 
denatured and reduced in 4M guanidine-HCl, 0.5M Tris-HCl. pH 8.6 and 0.7% 
.beta.-mercaptoethanol overnight. Iodoacetamide was added to the solution. 
The reaction was carried out in the dark for one hour and stopped by 
adding TFA to 0.1%. The reaction mixture was injected onto the HPLC column 
and the carboxyamidomethylated carrier protein subunit recovered as before 
and used for N-terminal molecule analysis. That analysis showed the same 
N-terminal amino acid molecule described in the example 1. 
EXAMPLE 3 
The carrier protein subunit was purified as in Example 2 through the gel 
filtration step (c). A 30 mg aliquot of the resulting sample containing SM 
binding activity was dissolved in 0.1% TFA solution and injected into a 
preparative Vydak C4 RP column. The column was eluted with a 0-60% 
acetonitrile gradient in 60 minutes. The SM binding activity peak which 
eluted at approximately 39% acetonitrile was collected and lyophilized. 
The SM binding activity was recovered quantitatively. 
The sample was subsequently resuspended in a Tris-glycine buffer containing 
.beta.-mercaptoethanol and separated by SDS-PAGE (12.5% polyacrylamide). 
Bands corresponding to 15, 21, 26, and 30 kDa carrier protein subunits 
(each of which bound labelled SM in a Western blot) were cut from the gel, 
and the proteins were electroeluted into Tris-glycine buffer. Each of the 
carrier protein subunits was lyophilized: recoveries were quantitative. 
EXAMPLE 4 
Experiments designed to measure the potential of SM carrier protein 
subunits to potentiate wound healing were carried out in the following 
manner Each of 6 anesthetized 300 gram male Sprague-Dawley rats was 
implanted subcutaneously (s.c.) with Schilling-Hunt wire mesh wound 
cylinders in each of the 4 quadrants on their back. Cylindrical chambers. 
20.times.5.8 mm i.d. with a volume of 520 ul, were constructed out of 
stainless steel wire mesh. One end was sealed with wire mesh and the other 
with a silastic disk. After implantation, the typical progression of wound 
healing events occurred: thrombosis of blood vessels followed sequentially 
by migration through the wire mesh of polymorphonuclear leukocytes, 
macrophages and fibroblasts, with subsequent fibroplasia, collagen 
synthesis and angiogenesis. During this process, the wound fluid that 
collected in the hollow chamber could be sampled or injected with active 
agents (s.c. through the silastic disk). Most of the healing was complete 
by 17 days after implantation: however, the central cavity was never 
completely obliterated. 
The 15 kDa SM carrier protein subunit was dissolved in PBS (150 mM sodium 
chloride, 10 mM sodium phosphate, pH 7.4). containing 0.1% bovine albumin. 
The wound chambers were injected with 100 ul of this solution (containing 
1.4 ug of the 15 kDa species) every 12 hours. This amount was selected to 
be only slightly in excess of the amount of biologically active 
somatomedins and thereby increase the half-life of somatomedins present. 
After 17 days, wound cylinders were removed, and the fibrous tissue was 
scraped carefully from each cylinder. Cylinders injected with 15 kDa 
carrier protein subunit material were all filled with dense fibrous tissue 
that was considerably greater than that in the controls. Specifically 19.5 
.+-.7 (SD) mg of protein were deposited in wound chambers containing 15 
kDa carrier protein subunit as compared to 7.0 .+-.1.6 mg deposited in 
controls. DNA synthesis was also much greater in carrier protein 
subunit-containing chambers (1160 .+-.200 ug vs 380 .+-.15 ug in 
controls). Likewise, hydroxyproline levels (an indicator of collagen 
synthesis) were significantly higher in carrier protein subunitcontaining 
chambers (460 ug vs 270 ug in controls). 
These results demonstrate that injection of 15 kDa carrier protein subunit 
into wound chambers markedly augments the rate of healing. 
EXAMPLE 5 
An animal experiment was conducted to show that the carrier protein 
subunits increase the serum half-life of SM-C. The 15 kDa human carrier 
protein subunit was shown to prolong the half-life of purified human SM-C 
injected into a rat's bloodstream. 
The complex between the 15 kDa carrier protein subunit and .sup.125 I-SM-C 
was formed by incubating .sup.125 I-SM-C with the carrier protein subunit 
overnight at 4.degree. C. in PBS (10 mM sodium phosphate, pH 7.25, 150 mM 
sodium chloride). The complex was separated from free .sup.125 I-SM-C by 
gel filtration. Specific activity of the .sup.125 I-SM-C was 
6.7.times.10.sup.5 cpm per ug. 
Rats (about 200 grams) were anesthetized and catheterized through the 
jugular vein. Prior to injections, the catheters and syringes were rinsed 
with 4% BSA (bovine serum albumin) to prevent sticking of the proteins to 
plastic surfaces. Four rats received BSA, four rats received 2 ug .sup.125 
I-SM-C alone, and four rats received 2 ug .sup.125 I-SM-C complexed with 
15 kDa carrier protein subunit. Both the complex and the SM-C were in PBS. 
One rat received 1 ug .sup.125 I-SM-C complexed with the carrier protein 
subunit. Blood samples (100-200 ul) were removed at multiple time points 
post injection. Blood cells were immediately separated from the plasma by 
centrifugation. A 25 ul plasma aliquot was counted to determine the 
concentration of .sup.125 I-SM-C present and a 10 ul aliquot was run on a 
15% polyacrylamide-SDS gel to determine SM-C integrity. Injections were 
carried out over a two day period. Each morning 2 rats were injected with 
the complex and 2 rats with SM-C alone. On a third day, 4 control rats 
were injected with BSA. 
This study demonstrates that the 15 kDa carrier protein subunit 
significantly increases the half-life of SM-C in the circulation. An equal 
number of counts (i.e., 1.3.times.10.sup.5 cpm/ml rat blood) of SM-C was 
added to rats either alone or complexed with the carrier protein subunit. 
As shown in FIG. 9, a majority of free SM-C is rapidly removed from the 
circulation, whereas the carrier protein subunit protects SM-C from that 
removal. (Samples run on 15% SDS [sodium dodecyl sulfate]polyacrylamide 
gels indicated that all .sup.125 I counts were SM-C; that is, there is no 
free .sup.125 I interfering with the experiment.) The continued appearance 
of the residual amount of free SM-C after 7.5 minutes may be due to SM-C 
occupying unsaturated rat carrier protein subunit molecules. Obviously, 
there were not sufficient endogenous carrier protein subunits to bind even 
30% of all the free SM-C injected. It should be noted that there are not 
sufficient endogenous unsaturated carrier protein subunits in rats or in 
humans to be therapeutically useful. Thus, SM-C must be administered 
complexed to its carrier protein subunit. 
RECOMBINANT DNA AND CARRIER PROTEIN-LIKE POLYPEPTIDES 
Preparation Of Oligonucleotide Probes Based On Protein Sequence Information 
The carrier protein contains subunits that may be isolated and retain the 
capability of binding somatomedins, including subunits having apparent 
molecular weights, if glycosylated, of about 15, 21, 26, 30 and 45 kDa, 
and significantly less, if not glycosylated or subjected to other post 
translation modifications. If the N-terminal sequences of the subunits are 
the same, and the various subunits are encoded by the same gene or genes, 
then it should be possible to prepare a probe based on a common N-terminal 
sequence to identify DNA coding for carrier protein-like polypeptides. A 
carrier protein subunit was isolated and purified as described in Example 
2, identified as S-15. The protein. S-15, was carboxymethylated and 
subjected to N-terminal sequence analysis using an Applied Biosystems Gas 
Phase Protein Sequencer, Model 470, by automated Edman degradation. The 
first 42 amino acids are in FIG. 1. In addition, the subunit S-15 was 
cleaved with the protease trypsin which specifically cleaves after 
arginines and lysines, unless lysine is followed by proline. Specifically, 
carboxymethylated S-15 was digested with trypsin in 0.3M sodium 
bicarbonate, pH 8.0. Tryptic fragments were separated by reverse phase 
HPLC using a Vydac C.sub.4 column. Purified fragments were collected and 
sequenced as described above. The sequences of several such tryptic 
fragments, denoted as T-1, T-6, T-7, T-1', and T-10, are also shown in 
FIG. 1. Due to the homology between the amino terminus and tryptic 
fragment T-7, it was determined that the first 57 N-terminal amino acids 
of subunit S-15, with two undetermined amino acids, are as shown in FIG. 
2a. 
Many oligonucleotides were designed from this molecule to serve as probes 
to screen cDNA libraries. These included short degenerate probes and long 
codon biased probes. One oligonucleotide corresponding to a portion of the 
N-terminal 57 amino acid molecule identified as the 48mer, is shown in 
FIG. 2a. 
Selection Of Tissues For Preparation Of PolyA.sup.+ FNA Containing Carrier 
Protein mRNA 
The strategy utilized to isolate carrier protein genes was to identify a 
tissue making large quantities of carrier protein, isolate mRNA from that 
tissue. construct a cDNA library from that mRNA, and screen for the gene 
using oligonucleotide probes. The hope was that an enriched cDNA library 
would contain more copies of such a gene than would a genomic (total DNA) 
library which will only contain perhaps one copy. There was no information 
in the literature to establish which tissue or cell type makes the carrier 
protein, a protein which is found in the serum. Fibroblasts had been shown 
to produce small amounts of a large but otherwise uncharacterized 
somatomedin binding protein (Adams. et al, supra). However, it is known 
that the majority of SM-C is synthesized in the liver. In addition. SM-C 
is synthesized by fibroblasts and other tissues such as the heart, bone, 
placenta, and kidney. Therefore, speculating that SM-C and the carrier 
protein would be synthesized by the same tissues, the liver and fibroblast 
cells were chosen as two potential sources of the mRNA coding for the 
carrier protein. 
In order to identify a tissue or cell line source of such mRNA, RNAs 
isolated from several human livers were prepared and tested for their 
ability to direct the synthesis of carrier protein. In addition, various 
fibroblast cell lines were assayed for their ability to make carrier 
protein. 
Preparation of PolyA.sup.+ Containing RNA 
Total and polyA.sup.+ containing RNA were isolated from various liver 
tissues and fibroblast cells according to standard procedures (Chirgwin, 
J.M., Pryzbyla, A.E., MacDonald, R.J. & Rutter, W.J. (1979) Biochemistry 
18, 5294-5299 and Iversen. P.L., Mata. J.E. & Hines, R.N. (1987) 
BioTechniques -5, 521-523.). Either tissue (e.g., liver) or cells (e.g., 
fibroblasts) were homogenized in GIT buffer (4M guanidinium 
isothiocyanate, 20 mM EDTA. 100 mM Tris-HCl, pH 7.6). Debris was removed, 
and the RNA-containing supernatant was brought to 2% Sarkosyl (sodium 
laurel sarkosinate) and 1%.beta.-mercaptoethanol. The mixture was then 
centrifuged through a cesium chloride gradient. Pellets were resuspended 
and extracted with phenol and chloroform and subsequently precipitated 
with ethanol. PolyA.sup.+ RNA, which represents the MRNA, was purified 
from total RNA by passing total RNA over an oligo-dT cellulose column 
(Aviv. H. & Leder, P. [1972]PNAS 69:1408). The resulting polyA.sup.+ 
containing RNA was eluted from the column with 10 mM Tris. pH 7.4, 1 mM 
EDTA, 0.05% sodium dodecyl sulfate (SDS), concentrated, and stored for 
further use. The liver polyA.sup.+ RNAs were assigned the names H10 and 
H14, indicative of the liver sample from which they were purified, and the 
fibroblast cell polyA.sup.+ RNAs assigned the code name W138, HS27, MRC5, 
8387, and MDA-MB-231 indicative of the cell source of the RNA. 
Testing Of RNA For Translation Products 
An aliquot of human liver polyA.sup.+ RNA from H10 and H14 were translated 
in vitro using a rabbit reticulocyte translation kit with .sup.35 
S-methionine according to standard procedures (Davis. L. G., et al., 
"Basic Methods in Molecular Biology." (Elsevier. New York, NY, 1986)). The 
protein translation products were immunoprecipitated (according to Davis) 
with an antibody provided by Robert C. Baxter (Royal Prince Alfred 
Hospital, Australia), prepared in accord with Martin. J.L., et al. 
"Antibody Against Acid-Stable Insulin-like Growth Factor Binding 
Protein...", J. Clin. Endocrinol. Metab., 261, pp. 799-801 (1985). That 
antibody was raised against material containing the so-called acid-stable 
subunit of the carrier protein obtained from human serum. 
Immunoprecipitated proteins were analyzed by SDS-polyacrylamide gel 
electrophoresis. Protein bands of about 68,000, 43,000, 39,000 and 32.000 
daltons were identified that reacted specifically with anti-carrier 
protein subunit antibody. The proteins were not precipitated by a control 
serum, which did not contain anti-carrier protein subunit antibodies. This 
result suggested that carrier protein is being made by a liver and that a 
cDNA library made from liver mRNA should contain the carrier protein gene. 
Several fibroblast cell lines were also tested for their ability to produce 
the carrier protein. For example. WI38 embryonic fibroblasts (American 
Type Culture Collection No. CCL-75) were grown to 70-80% confluence in 
DMEM-F12 media containing 10% fetal calf serum. Cells were switched to 
serum free media and incubated for 72 hours. Culture supernatants were 
harvested and concentrated by TCA precipitation or by centrifugation. 
Samples were subjected to SM-Western analysis (SDS-PAGE step being carried 
out under non-reducing conditions) which demonstrated that WI38 cells 
synthesized and secreted at least 4 proteins capable of binding SM-C. in 
the size range of 25,000-45,000 daltons. Of these, an about 40,000 dalton 
protein (by reducing SDS-PAGE) was also specifically recognized by the 
anti-carrier protein subunit antibody. In this experiment, the 72 hour 
incubation of WI38 cells in serum free medium included the addition of 
.sup.35 S-cysteine. The proteins were immunoprecipitated with anti-carrier 
protein subunit antibody and analyzed by SDS-PAGE under reducing 
conditions. 
Other cell lines encoding carrier protein subunits that were both 
recognized by anti-carrier protein subunit antibody and bound by SM-C 
include HS27 (human fibroblast), MRC5 (human fibroblast), 8387 (human 
fibrosarcoma), and MDA-MB-231 (human breast cancer). It is expected that 
polyA.sup.+ RNA isolated from other fibroblast lines would also encode 
carrier protein. 
It should be recognized that the polyA.sup.+ RNA product obtained from 
these sources contain a very large number of different mRNAs. Except for 
the mRNA specific for carrier protein or carrier protein subunits, the 
other mRNAs are undesirable contaminants. Unfortunately, these contaminant 
RNAs may behave similarly to carrier protein subunit mRNA throughout the 
remainder of the cloning process of this invention. Therefore, their 
presence in the polyA RNA will result in the ultimate preparation of a 
large number of unwanted bacterial clones, which contain genes that may 
code for polypeptides other than carrier protein. This contamination 
presents complex screening problems in the isolation of the desired 
carrier protein hybrid clones. In the case of carrier protein, the 
screening problem was further exacerbated by the lack of a sufficiently 
purified sample of carrier protein mRNA or DNA. or portion thereof, to act 
as a screening probe for the identification of the desired clones. The 
only available probes were those based on the limited N-terminal protein 
molecule information. Therefore, the screening process for the carrier 
protein clones is very time-consuming and difficult. Furthermore, because 
only a very small percentage of carrier protein clones themselves are 
expected to express carrier protein-like polypeptide in a biologically or 
immunologically active form, the isolation of an active clone is a 
difficult screening process. 
Synthesis Of Double Stranded cDNA Containing Carrier Protein cDNA 
PolyA.sup.+ RNA containing carrier protein mRNA was used as a template to 
prepare complementary DNA ("cDNA"), essentially as described by Gubler and 
Hoffman. cDNA libraries were made from the mRNAs which had been shown to 
encode potential carrier protein-like polypeptides. The libraries were 
constructed in the .lambda. vector gt10, but could be constructed in other 
vectors as well (e.g., .lambda. gt11 [Young, R.A. & Davis, R.W. (1983) 
Proc. Natl. Acad. Sci. USA 80, 1194-1198]). Double-stranded cDNA was 
generated essentially according to the Gubler-Hoffman method (Gubler, U. & 
Hoffman. B.J. (1983) Gene 25, 263-269). In this protocol, first strand 
cDNA was synthesized using Moloney Reverse Transcriptase to copy the 
polyA.sup.+ RNA. Libraries described below include a random-primed human 
liver cDNA library (H14), two oligo-dT-primed human liver cDNA libraries 
(H14, H10/H14 [a pool of H10 and H14]). and an oligo-dT-primed human 
embryonic fibroblast library (WI38). Random primers (pd(N).sub.6) and 
oligo-dT (pT.sub.12 -18) primers were obtained from Pharmacia. The second 
strand was produced using a combination of RNAseH and DNA polymerase I. 
The resulting cDNA population is in fact a complex mixture of cDNAs 
originating from the different mRNAs, which were present in the 
polyA.sup.+ RNA. In addition, because of premature termination by Moloney 
reverse transcriptase, many of the cDNAs are incomplete copies of the 
various mRNAs in the polyA.sup.+ mRNA. 
Cloning Of Double-Stranded cDNA 
A wide variety of host vehicle combinations may be employed in cloning or 
expressing the double-stranded cDNA prepared in accordance with this 
invention. For example, useful cloning or expression vehicles may consist 
of segments of chromosomal, non-chromosomal and synthetic DNA molecules, 
such as various known derivatives of SV40 and known bacterial plasmids, 
e.g., plasmids from E. coli including col El, pCRl, pBR322, pMB9 and their 
derivatives, wider host range plasmids, e.g., RP4. phage DNAs. e.g., the 
numerous derivatives of phage .lambda., e.g., NM 989, and other DNA 
phages. e.g., M13 and Filamenteous single stranded DNA phages and vectors 
derived from combinations of plasmids and phage DNAs such as plasmids 
which have been modified to employ phage DNA or other expression control 
molecules or yeast plasmids such as the 2 .mu.plasmid or derivatives 
thereof. Useful cloning or expression hosts may include bacterial hosts 
such as E. coli HB 101, E. coli X1776, E. coli X2282, E. coli MRCI, E. 
coli LE392, E. coli C600 and strains of Pseudomonas, Bacillus subtilis, 
Bacillus stearothermophilus and other bacteria, yeasts and other fungi, 
animal, insect or plant cells. Of course, not all host/vector combinations 
may be equally efficient. The particular selection of host vehicle 
combination may be made by those of skill in the art after due 
consideration of the principles set forth herein without departing from 
the scope of this invention. 
Furthermore, within each specific cloning or expression vehicle, various 
sites may be selected for insertion of the double-stranded DNA. These 
sites are usually designated by the restriction endonuclease which cuts 
them. These sites are well recognized by those of skill in the art. It is, 
of course, to be understood that a cloning or expression vehicle useful in 
this invention need not have a restriction endonuclease site for insertion 
of the chosen DNA fragment. Instead, the vehicle could be joined to the 
fragment by alternative means. 
The cloning or expression vehicle or vector, and in particular the site 
chosen therein for attachment of a selected DNA fragment to form a 
recombinant DNA molecule, is determined by a variety of factors. e.g., 
number of sites susceptible to a particular restriction enzyme, size of 
the protein to be expressed, susceptibility of the desired protein to 
proteolytic degradation by host cell enzymes, contamination or binding of 
the protein to be expressed by host cell proteins difficult to remove 
during purification, expression characteristics, such as the location of 
start and stop codons relative to the vector molecules, and other factors 
recognized by those of skill in the art. The choice of a vector and an 
insertion site for a particular gene is determined by a balance of these 
factors, not all selections being equally effective for a given case. 
Although several methods are known in the art for inserting foreign DNA 
into a cloning vehicle or expression vector to form a recombinant DNA 
molecule, the method preferred for initial cloning in accordance with this 
invention is digesting .lambda. gt10 with EcoRI. The double-stranded cDNA 
is then ligated to this .lambda. gt10 DNA, after first adding EcoRI 
linkers to the cDNA molecules. The resulting recombinant DNA molecule now 
carries an inserted gene at the chosen position in the cloning vector. 
Of course, other known methods of inserting DNA molecules into cloning or 
expression vehicles to form recombinant DNA molecules are equally useful 
in this invention. These include, for example, dA dT tailing, direct 
ligation, synthetic linkers, exonuclease and polymerase linked repair 
reactions followed by ligation, or extension of the DNA strand with DNA 
polymerase and an appropriate single-stranded template followed by 
ligation. 
It should, of course, be understood that the nucleotide molecules of cDNA 
fragments inserted at the selected site of the cloning vehicle may include 
nucleotides which are not part of the actual gene coding for the desired 
polypeptide or may include only a fragment of the complete gene for the 
desired protein. It is only required that whatever DNA molecule is finally 
inserted, a transformed host will produce a polypeptide having a 
somatomedin regulating biological activity of the carrier protein and 
being capable of binding somatomedin-like polypeptides, or that the DNA 
molecule itself is of use as a hybridization probe to select clones which 
contain DNA molecules useful in the production of polypeptides having such 
biological and binding activity. 
The cloning vehicle or expression vector containing the foreign gene is 
employed to transform a host so as to permit that host to express carrier 
protein-like polypeptides. The selection of an appropriate host is also 
controlled by a number of factors recognized by the art. These include, 
for example, compatibility with the chosen vector, toxicity of proteins 
encoded by the hybrid plasmid, ease of recovery of the desired protein, 
expression characteristics, safety and cost. A balance of these factors 
must be struck with the understanding that not all hosts may be equally 
effective for either the cloning or expression of a particular recombinant 
DNA molecule. 
In the present synthesis, the preferred initial cloning vehicle is .lambda. 
gt10 and the preferred initial restriction endonuclease site is EcoRI. The 
preferred initial host is E. coli. 
EcoR1-restricted .lambda. gt10 DNA (Promega) was ligated to EcoR1 linkered 
cDNA molecules prepared as described in Maniatis, T., et al., Molecular 
Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory. Cold Spring 
Harbor, NY, 1982) and Davis, L. G., et al., "Basic Methods in Molecular 
Biology." (Elsevier, New York, NY 1986) ("Maniatis"). 
The hybrid DNA obtained after annealing is. of course, a large mixture of 
different recombinant DNA molecules and some cloning vehicles without 
inserted DNA molecules. However, each recombinant DNA molecule contains a 
cDNA segment at the EcoRI site. Each such cDNA segment may comprise a gene 
or a fragment thereof. Only a very few of the cDNA fragments code for 
carrier protein or a portion thereof. The vast majority code for one of 
the other proteins or portions thereof whose mRNAs were part of the 
polyA.sup.+ RNA used in the process of this invention. It should also be 
understood that it is possible that none of the clones of the 
above-prepared library may permit the expression of carrier protein-like 
polypeptides. Instead, they may only be useful in screening for and 
identifying such a clone. 
The resultant .lambda. DNA vectors containing cDNA inserts were packaged 
into .lambda. phage using a .lambda. phage packaging kit (Stratagene). 
E. coli cells (e.g., C600 hfl) were infected with the recombinant phage and 
plated onto enriched media plates. (e.g. LB). Plates were incubated at 
37.degree. C. until phage plaques were visible. 
The phage plaques (clones) contain a variety of recombinant DNA molecules 
representing sized, complete or partial copies of the mixture of 
polyA.sup.+ RNA obtained from the liver. Each of the majority of these 
plaques will contain a single recombinant DNA molecule. However, only a 
very few of these recombinant DNA molecules are related to carrier 
protein. Accordingly, the clones must be screened to select the carrier 
protein related clones from the others. 
Screening For A Clone Containing Carrier Protein cDNA 
There are several approaches to screen for clones containing carrier 
protein cDNA. These include, for example, RNA selection hybridization, 
differential hybridization; hybridization with a synthetic probe or 
screening for clones that produce the desired protein by immunological or 
biological assays. We have chosen hybridization with a synthetic probe as 
being the most convenient and promising method for primary clone 
screening. 
There is no assurance that the recombinant DNA molecules and bacterial 
cultures transformed therewith, which are identified by hybridization with 
a probe, contain the complete carrier protein cDNA molecule or that the 
DNA molecule actually codes for carrier protein or will permit the clone 
to express a carrier protein-like polypeptide. However, the recombinant 
DNA molecules will certainly contain extensive nucleotide molecules 
complementary to the carrier protein subunit mRNA coding molecule. 
Therefore, the recombinant DNA molecule may at least be used as a source 
of a probe to screen rapidly other recombinant DNA molecules and clones 
transformed with them to identify further sets of clones which will 
contain an authentic or complete carrier protein subunit nucleotide coding 
molecule. These clones may then be analyzed directly for possible 
expression of polypeptides displaying the biological and binding activity 
of carrier protein. More importantly, the nucleotide molecule of the 
inserted DNA fragment of these hybrid plasmids and its amino acid 
translation product may be determined using conventional means and that 
DNA molecule used to construct appropriate expression vectors that permit 
the synthesis of carrier protein-like polypeptides in appropriate hosts 
transformed with them. 
Oligonucleotide Probe Hybridization 
The phage cDNA library was mixed with E. coli and plated onto LB (enriched 
media) plates. The plates were incubated at 37.degree. C. until phage 
plaques were visible. Each plaque represents a clone of a unique .lambda. 
gt10 phage containing a cDNA insert. Approximately 0.5-1.0 million phage 
plaques were analyzed per experiment. 
Analysis was carried out by transferring the phage DNA of these plaques 
from the plates onto nitrocellulose filters (0.45 um pore diameter 
Schleicher and Schuell or Millipore), using standard techniques (Davis and 
Maniatis). Thus, the DNA pattern on the filter was a replica of the plaque 
pattern on the plate. After identification of inserts contained within 
phage DNA that hybridized to the probe, the filters can be matched with 
the plates and phage isolated. 
An oligonucleotide probe, the 48mer, of 48 bases (shown in FIG. 2a) was 
used to screen the random-primed human liver cDNA library H14. The probe 
corresponded to the molecule spanning the nucleotides encoding amino acids 
Ala[29]through Leu[44]of the carrier protein subunit S-15. This single 
oligonucleotide was designed to maximize on bias for human codons. 
Hybridization conditions were determined by binding the 48-base probe 
(48mer) to Southern blots of human genomic DNA from the placenta and of a 
181-bp synthetic DNA encoding amino acids Gly[1]through Tyr[57](shown in 
FIG. 2b) under different degrees of stringency. The final conditions for 
hybridization, which would allow for gene identification with minimal 
background, was 40% formamide, 5X SSPE (0.9M sodium chloride, 50 mM sodium 
phosphate, pH 7.4, 5 mM EDTA). 42.degree. C. 
Nitrocellulose filters containing replicas of the phage plaques from the 
random-primed H14 human liver cDNA library were hybridized with .sup.32 
P-labelled 48mer using the hybridization conditions described above. 
Hybridization was usually carried out overnight, and the filters were 
rinsed several times in 0.1X SSC (15 mM sodium chloride. 1.5 mM sodium 
citrate, pH 7.0). 0.1% SDS at 45.degree.-50.degree. C. prior to 
autoradiography. DNA's that hybridized strongly to the 48mer were 
identified by autoradiography and the corresponding phage plaques were 
isolated. Since the original plating of phage was done at a high density, 
a second round of plating and screening was required to isolate single 
plaques. This second round of screening also verified that the original 
isolated phage plaques did indeed hybridize to the 48mer. Single plaques 
were picked from the plates and the phage were allowed to elute into phage 
buffer (100 mM NaCl. 10 mM MgSO.sub.4, 50 mM Tris. pH 7.5, 0.01% gelatin). 
After removing the bacteria by centrifugation, these phage stocks were 
maintained at 4.degree. C. Phage DNA was purified and characterized (i.e., 
restricted by restriction endonucleases such as EcoRI in order to 
determine insert size) following standard procedures (e.g., Maniatis). 
Inserts were frequently subcloned into smaller plasmids, such as pBR322 or 
pGEM, at the EcoRI site, using standard procedures. 
A number of positive plaques were identified (48 per 600,000 plaques 
screened). Of these, 9 were chosen for further analysis. Two of these 
clones (designated cLCP 0.70 and cLCP 0.77), which were approximately 700 
to 800 bp in size and which showed the most intense binding by the 48mer 
probe were cut into smaller fragments prior to sequencing. 
Fragments hybridizing to the 48mer, which would be initial sequencing 
candidates were identified in the following manner, cDNA inserts LCP 0.70 
and LCP 0.77 were cleaved with restriction endonuclease HaeIII. These 
fragments were separated by agarose gel electrophoresis, transferred to a 
nitrocellulose membrane, and probed with .sup.32 P-labelled 48mer probe. 
When HaeIII fragments were probed, only one fragment bound the 48mer. This 
90 bp fragment was present in both clones LCP 0.70 and LCP 0.77. It was 
isolated and sequenced according to Sanger, F. et al., Proc. Natl. Acad. 
Sci., 74, p. 5463 (1977). The DNA molecules of the 90 bp fragments from 
both LCP 0.70 and LCP 0.77 corresponded exactly to the carrier protein 
subunit. S-15, amino acid sequence spanning Gln[23]through Glu[50], as 
shown below. The top line represents the first 57 amino acids of the 
carrier protein subunit, S-15, and the bottom line represents the 
translation of the 84 bp HaeIII fragments. The one non-match is the result 
of the fact that the amino acid at position 45 was unidentified. DNA 
molecule analysis identified it as a threonine (T). 
##STR3## 
These clones were designated as cLCP 0.70 and cLCP 0.77. their recombinant 
DNA molecules as .lambda. gtlO:LCP 0.70 and .lambda. gt10:LCP 0.77, and 
their DNA inserts LCP 0.70 and LCP 0.77. This nomenclature indicates that 
the clone and recombinant DNA molecule comprises phage .lambda. gt10, 
containing carrier protein related cDNA isolated from liver cDNA. 
Inserts LCP 0.70 and LCP 0.77 were shown to be similar in size and 
restriction sites. Inserts LCP 0.70 and LCP 0.77 are approximately 700 and 
770 bp. respectively. The restriction maps of LCP 0.70 and LCP 0.77 are 
shown in FIG. 3a. The DNA sequences of the LCP 0.70 and LCP 0.77 inserts, 
obtained by both single and double-stranded dideoxy-sequencing (Sanger, 
F., et al., Proc Natl Acad Sci USA 74, 5463 (1977)), are included in the 
sequence shown in FIG. 4, nucleotides 1-699 and 7-769, respectively. In 
addition to the amino terminus, tryptic fragments T1' and T10 corresponded 
to the DNA molecules of these clones, LCP 0.70 and LCP 0.77 are 
sufficiently large to encode 17.558 and 20,320 dalton proteins, 
respectively. Thus, the information required to encode the entire S-15 
molecule is contained within these inserts. 
Identification Of Clones Containing DNA Sequences Coding For Carrier 
Protein By Cross-Hybridizing To Either LCP 0.70 and LCP 0.77 
The recombinant DNA molecules and DNA inserts of clones cLCP 0.70 and cLCP 
0.77 isolated as described above, were used to screen the library of 
clones previously prepared from cDNA by hybridization to phage plaques. 
This method allows rapid identification of related clones by hybridization 
of a radioactive probe made from LCP 0.70 to the DNA of recombinant phage 
fixed on nitrocellulose filters. 
Nitrocellulose filters containing phage DNAs that corresponded to phage 
plaques transferred from LB plates were prepared as described above. 
Either the 700 bp LCP 0.70 or the 770 bp LCP 0.77 EcoRI restriction 
fragment was used to screen human liver random-primed cDNA library H14, 
human liver oligo-dT-primed cDNA library HlO/H14, and human embryonic 
fibroblast oligo-dT-primed cDNA library WI38. These probes could also be 
used to screen other cDNA libraries constructed using RNAs from other 
tissues encoding the carrier protein. In addition they could be used to 
screen genomic libraries. 
The probe fragment (LCP 0.70 or LCP 0.77) was purified by electrophoresis 
of the EcqRI digestion products of the recombinant DNA molecules (to 
separate the insert from the cloning vehicle) in about a 1% agarose gel 
followed by electroelution onto DE81 paper. The specific fragment was then 
concentrated and .sup.32 P-labelled by "nick translation" by standard 
procedures. 
Hybridization of the above probe to the nitrocellulose filter containing 
the cDNA clones was carried out essentially as described above. 
About 500,000 clones originating from oligo-dT-primed human liver cDNA 
library H10/H14 and about 500,000 clones originating from oligo-dT-primed 
human embryonic fibroblast cDNA library WI38 were screened. 
The frequency of positive signals in the WI38 fibroblast library was 
approximately 0.1%, whereas the frequency in the liver libraries was only 
0.01-0.02%. Positive clones were plaque-purified and characterized by 
restriction mapping and sequence analysis to identify other clones 
containing carrier protein cDNA. Clones were sequenced using single- and 
double-stranded sequencing techniques (Sanger). 
A clone containing a 2.3 kb insert (cLCP 2.3) was isolated from human liver 
oligo dT-primed cDNA library H10/H14 which contains that full-length 
carrier protein-like coding sequence. Clones containing inserts of 1.8 kb 
(cFCP 1.8) and 2.5 kb (cFCP 2.5), respectively were isolated from the WI38 
fibroblast oligo-dT-primed cDNA library. DNA sequence analysis of the 
clones (FIG. 4) showed that both contain the entire carrier protein-like 
polypeptide coding sequence. The encoded protein consists of a 27 amino 
acid (81 nucleotide) leader plus a 264 amino acid (792 nucleotide) mature 
coding region. Both the liver and fibroblast clones display essentially 
the same nucleotide sequence in the coding region. One of the liver clones 
encodes a GLY instead of an ALA at amino acid position 5, where position 1 
is the first amino acid of the mature protein. This polymorphism 
corresponds to that observed in carrier protein subunits purified from 
Cohn fraction IV-1. 
Northern analysis of WI38 human embryonic fibroblast RNA, human liver RNAs 
HlO/H14. human placenta RNA, and macaque liver RNA using LCP 0.70 or LCP 
0.77 as a probe indicated that the carrier protein mRNA is approximately 
2,000-2,500 bases in size. Thus, the 2.2-2.4 kb clones likely represent 
full-length cDNAs corresponding to those RNAs. Analysis of the human liver 
cDNA library and clone cLCP 2.3 by polymerase chain reaction (PCR) 
amplification (Saiki, R. K., et al. Science 239, pp. 487-491 [1988]) 
suggests that cLCP 2.3 may have a small deletion of approximately 200 bp 
in the 3' untranslated region. In fact, recently a clone containing a 2.5 
kb insert (cLCP 2.5) was isolated from the liver cDNA library. This insert 
(LCP 2.5) is the same as LCP 2.3 except for a 200-bp "insertion" between 
the XhoI site at 1063 and the SphI site at 1270 (FIG. 3b). LCP 2.5 is 
apparently analogous to FCP 2.5. 
It is, of course, evident that this method of clone screening using the DNA 
insert of clones LCP 0.70 and LCP 0.77, as described above, may be 
employed equally well on other clones containing DNA molecules arising 
from recombinant DNA technology. synthesis, natural sources or a 
combination thereof and on clones containing DNA molecules related to any 
of the above DNA molecules by mutation, including single or multiple, base 
substitutions, insertions, inversions, or deletions. Therefore, such DNA 
molecules and their identification also fall within this invention. It is 
also to be understood that DNA molecules, which are not screened by the 
above DNA molecule, yet which as a result of their arrangement of 
nucleotides code for the polypeptides coded for by the above DNA molecules 
also fall within this invention. 
In addition, because of the expected homology between the DNA molecule 
coding for human carrier protein-like polypeptide and the DNA molecule 
coding for carrier proteins from non-human sources, the DNA molecules of 
this invention are useful in the selection of the DNA coding for those 
non-human carrier proteins and in the cloning and expression of those 
non-human carrier proteins for use in therapeutic compositions and 
methods. Finally, the DNA molecules of this invention or oligo-nucleotides 
prepared and derived from them may be employed to select other DNA 
molecules that encode carrier protein-like polypeptides that may not be 
the carrier protein or a carrier protein subunit. Those molecules and 
polypeptides are also part of this invention. 
Expression Of Polypeptides Displaying An Activity Of The Carrier Protein 
Production of polypeptides by expressing DNA molecules encoding a carrier 
protein-like polypeptide was carried out in E. coli and mammalian cells. 
Expression in E. coli of Full-Length Carrier Protein-Like Sequence With 
Alternate Signal Sequence 
A DNA fragment containing the entire coding region of the carrier protein 
gene in which the gene's signal sequence was replaced by that for 
preproinsulin was ligated into the expression vector pKK233-2 (Pharmacia). 
This vector contains a trp-lac fusion promoter in which the -35 trp signal 
is placed 17 bases (the consensus distance) from the lac -10 region. The 
presence of the lac operator sequences allows expression from this 
promoter to be induced by adding IPTG 
(isopropyl-.beta.-D-thiogalactopyranoside) to the medium. In addition, 
this vector contains the lacZ ribosome binding site. 
The insert (pDJ4219) containing the preproinsulin signal sequence fused to 
the carrier protein gene's mature coding sequence was accomplished in the 
following manner (shown in FIG. 5). A preproinsulin signal sequence was 
synthesized in which the initiating ATG was contained within an NcoI 
restriction site. The signal sequence was followed by the nucleotides 
GGCGCGAGCTCG encoding the first four amino acids of the mature carrier 
protein, through the SacI site. Thus, it was possible to generate the 
NcoI/SacI fragment shown in FIG. 5. This fragment was ligated to the 
SacI/XhoI fragment containing the rest of the coding sequence for the 
carrier protein, also shown in FIG. 5. The XhoI site, which is located 85 
bp beyond the translation termination site, had been converted to a 
HindIII site by the addition of HindIII linkers using standard procedures. 
The resulting NcoI/HindIII fragment containing the preproinsulin signal 
sequence and the carrier protein coding region was inserted into the NcoI 
and HindIII sites of pKK233-2. Expression of this construction in E. coli 
induced by IPTG yielded a 25,000-30,000 dalton protein, identified by its 
ability to bind anti-carrier protein antibody. Expression was carried out 
in the presence of .sup.35 S-cysteine. Two hours after induction by IPTG, 
the cell extract (cytoplasm and periplasmic space) was immunoprecipitated 
with anti-carrier protein antibody and submitted to SDS-PAGE. The ability 
of the carrier protein to be induced by IPTG was demonstrated, since cells 
containing this construction grown in the absence of IPTG induction 
expressed only very small quantities of the 25,000-30,000 dalton protein. 
Controls in which pKK233-2 alone was tested showed no protein in this size 
range. 
Expression In COS Cells Of A Partial Carrier Protein-Like Sequence 
Insert fragments from pDJ4209 and pDJ4211 (shown in FIG. 6) were ligated 
into mammalian expression vector pSVL or pDJ4210 (Pharmacia) at the unique 
XbaI site, pSVL contains the SV40 late promoter, intron, and 
polyadenylation site. It also has SV40 and pBR322 origins of replication, 
pDJ4210 is similar to pSVL but contains the origin of replication from 
pUC19 instead of pBR322. 
Each of these inserts contains a partial carrier protein gene, specifically 
the first 120 codons of the mature sequence followed by a synthetic 
sequence (5'-CTCTAGAG.,3') which terminates the reading frame. Each has a 
different control region. 
pDJ4209 contains the entire 5' untranslated region (114 nucleotides) 
stretching from the EcoRI site, which has been converted to an XbaI site. 
It also contains the carrier protein signal sequence. The pDJ4209 XbaI 
fragment contained in pSVL is called pDJ4207. 
pDJ4211 contains a 44 nucleotide 5' untranslated region and the carrier 
protein signal sequence. The pDJ4211 Xbal fragment contained in pDJ4210 is 
called pDJ4212 and is shown in FIG. 7. 
The vectors containing the partial carrier protein genes were transfected 
into COS cells (defective SV40 transformed simian cells) to measure 
transient expression. Cells were grown in DMEM-F12. Proteins were labelled 
with .sup.35 S-cysteine. Media was collected, immunoprecipitated with 
anti-carrier protein antibody. and submitted to SDS-PAGE. Expression 
studies using pDJ4212 and pDJ4207 yielded two proteins of approximately 
14,000 and 16,000 daltons. Expression of these proteins was greater with 
pDJ4212 than with pDJ4207. 
Expression In CHO Cells Of A Full-Length Carrier Protein-Like Sequence 
The 1.66 kb EcoRI/HindIII fragment of LCP 2.3 which contains the entire 
carrier protein gene plus 5' and 3' untranslated regions (114 and 700 
nucleotides, respectively) was inserted into mammalian expression vector 
pKG3226 which contains a .beta.-actin promoter (licensed from Stanford 
University) and other functions necessary for expression in mammalian 
cells. The resultant vector, called pKG4403 is shown in FIG. 8, pKG4403 
was transformed into CHO (Chinese hamster ovary) cells; stably transformed 
lines were established by drug selection. Serum-free conditioned media 
from the transformed CHO pool was analyzed for carrier protein-like 
polypeptide expression by immunoprecipitation of .sup.35 S-labelled 
products and by ability to bind .sup.1251 -SM-C in an SM-C Western. For 
detection by immunoprecipitation, cells were grown to 80% confluence in 
DMEM-F12 supplemented with 10% fetal bovine serum, switched to serum-free 
media, starved for cysteine 1 hour, and subsequently labelled overnight 
with .sup.35 S-cysteine. The media was immunoprecipitated with 
anti-carrier protein subunit antibody, and the resulting proteins were 
analyzed by SDS-PAGE under reducing conditions. Carrier protein-like 
polypeptides of 37,000 and 39,000 daltons were specifically identified. 
For detection by SM-C binding, serum-free conditioned media (unlabelled) 
was collected 48 hours after seeding the transformed pool and was 
subjected to SDS-PAGE under nonreducing conditions. The proteins were 
transferred from the gel to a nitrocellulose filter which was probed with 
.sup.125 I-SM-C. Two novel carrier protein-like polypeptides of 43,000 and 
45,000 daltons were observed. A 23,000 dalton protein endogenous to CHO 
cells was detected in the transformed pool as well as in the 
non-transformed control CHO pool. The size difference (37,000 and 39,000 
versus 43,000 and 45,000) is likely due to whether SDS-PAGE was conducted 
under reducing or non-reducing conditions. 
This gene of LCP 2.3 does not exclude the possibility that modifications to 
the gene such as mutations, including single or multiple, base 
substitutions, deletions, insertions, or inversions may not have already 
occurred in the gene or may not be employed subsequently to modify its 
properties or the properties of the polypeptides expressed therefrom. Nor 
does it exclude any polymorphism which may result in physiologically 
similar but structurally slightly different genes or polypeptides than 
that shown in FIG. 4. 
It should, of course, be understood that cloned cDNA from polyA.sup.+ RNA 
by the usual procedures may lack 5'-terminal nucleotides and may even 
contain artifactual molecules. 
The structure of the polypeptide depicted in FIG. 4, of course, does not 
take into account any modifications to the polypeptide caused by its 
interaction with in vivo enzymes. e.g., glycosylation. Therefore, it must 
be understood that the amino acid molecule depicted in FIG. 4 may not be 
identical with carrier protein produced in vivo. 
It should be understood that while the chromosomal gene encoding carrier 
protein activity may not be expressible in bacterial hosts because these 
intervening molecules may not be processed correctly by such hosts, the 
chromosomal genes are likely to be very useful in the production of 
carrier protein-like polypeptides in eukaryotic hosts where the human 
noncoding regions, introns and coding regions may be important for high 
levels of expression and correct processing of the product to biologically 
active carrier protein-like polypeptides. 
Improving The Yield And Activity Of Polypeptides Displayinq Carrier Protein 
Activity 
The level of production of a protein is governed by three major factors: 
the number of copies of its gene within the cell, the efficiency with 
which those gene copies are transcribed and the efficiency with which they 
are translated. Efficiency of transcription and translation (which 
together comprise expression) is in turn dependent upon nucleotide 
molecules, normally situated ahead of the desired coding molecule. These 
nucleotide molecules or expression control molecules define the location 
at which RNA polymerase interacts to initiate transcription (the promoter 
molecule) and at which ribosomes bind and interact with the mRNA (the 
product of transcription) to initiate translation. Not all such expression 
control molecules function with equal efficiency. It is thus of advantage 
to separate the specific coding molecules for the desired protein from 
their adjacent nucleotide molecules and to fuse them instead to other 
known expression control molecules so as to favor higher levels of 
expression. This having been achieved, the newly engineered DNA fragments 
may be inserted into higher copy number plasmids or bacteriophage 
derivatives in order to increase the number of gene copies within the cell 
and thereby further to improve the yield of expressed protein. 
Several expression control molecules may by employed as described above. 
These include the operator, promoter and ribosome binding and interaction 
molecules (including molecules such as the Shine-Dalgarno molecules) of 
the lactose operon of E. coli ("the lac system"). the corresponding 
molecules of the tryptophan synthetase system of E. coli ("the trp 
system"). the major operator and promoter regions of phage .lambda. 
(O.sub.L P.sub.L and O.sub.R P.sub.R), the bacteriophage T7 promoter 
recognized only be T7 RNA polymerase, a control region of Filamentous 
single-stranded DNA phages. SV40 early and late promoters, actin 
promoters, promoters located on the long terminal repeats of retroviruses, 
or other molecules which control the expression of genes of prokaryotic or 
eukaryotic cells and their viruses or combinations thereof. Therefore, to 
improve the production of a particular polypeptide in an appropriate host, 
the gene coding for that polypeptide may be prepared as before and 
inserted into a recombinant DNA molecule closer to its former expression 
control molecule or under the control of one of the above improved 
expression control molecules. Such methods are known in the art. 
Other methods to improve the efficiency of translation involve insertion of 
chemically or enzymatically prepared oligonucleotides in front of the 
initiating codon. By this procedure a more optimal primary and secondary 
structure of the messenger RNA can be obtained. More specifically, a 
molecule can be so designed that the initiating AUG codon occurs in a 
readily accessible position (i.e., not masked by secondary structure) 
either at the top of a hairpin or in other single-stranded regions. Also 
the position and molecule of the aforementioned Shine-Dalgarno segment can 
likewise be optimized. The importance of the general structure (folding) 
of the messenger RNA has been documented. 
Further increases in the cellular yield of the desired products depend upon 
an increase in the number of genes that can be utilized in the cell. This 
may be achieved by insertion of the carrier protein-like gene (with or 
without its transcription and translation control elements) in a higher 
copy number plasmid or in a temperature-controlled copy number plasmid 
(i.e., a plasmid which carries a mutation such that the copy number of the 
plasmid increases after shifting up the temperature. 
Alternatively, an increase in gene dosage can be achieved for example by 
insertion of recombinant DNA molecules engineered in the way described 
previously into the temperate bacteriophage, most simply by digestion of 
the plasmid with a restriction enzyme, to give a linear molecule which is 
then mixed with a restricted phage .lambda.cloning vehicle and the 
recombinant DNA molecule produced by incubation with DNA ligase. The 
desired recombinant phage is then selected as before and used to 
lysogenize a host strain of E. coli. 
Therefore, it should be understood that the insert DNA of this invention 
may be inserted into other expression vectors, as previously described 
(supra) and these vectors employed in various hosts, as previously 
described (supra) to improve the expression of the gene coding for carrier 
protein subunit. 
The biological activity of the carrier proteinlike polypeptides produced in 
accordance with this invention may also be improved by using the DNA 
molecules of this invention to transform mammalian cell systems and to 
express the gene in those systems. Such mammalian systems are known. One 
such system is the CHO (Chinese Hamster ovary) (DHFR.sup.-) cell system in 
which the gene expression may be amplified by methotrexate (MTX). These 
expression systems permit the production of glycosylated proteins. Such 
cells can be induced to greatly amplify the copy number of the carrier 
protein-like gene. 
It should also be understood that carrier protein-like polypeptides may 
also be prepared in the form of a fused protein (e.g., linked to a 
prokaryotic or eukaryotic N-terminal segment directing excretion). in the 
form of procarrier protein-like polypeptide (e.g., starting with all or 
parts of the carrier protein signal molecule which could be cleaved off 
upon excretion) or as a mature carrier protein-like polypeptide (by 
cleavage of any extraneous amino acids. including an initial methionine 
during expression and excretion) or in the form of a f-met-carrier 
protein-like polypeptide. One particularly useful polypeptide in 
accordance with this invention would be mature carrier-like polypeptide 
with an easily cleaved amino acid or series of amino acids attached to the 
amino terminus. Such constructions would allow synthesis of the protein in 
an appropriate host, where a start signal not present in mature carrier 
protein subunits is needed, and then cleavage of the extra amino acids to 
produce mature carrier protein subunits. 
When the carrier protein subunit or carrier protein-like polypeptide is to 
be used in combination with somatomedin-like molecules for therapy, the 
two molecules could be co-produced in the same cell, preferably in 
mammalian cells. Vectors containing both genes could be cotransformed and 
stable cell lines selected that expressed both proteins. Thus, only one 
fermentation and purification scheme would be required to produce the 
complex containing both carrier proteinlike and the somatomedin-like 
polypeptides. 
The yield of these different forms of polypeptide may be improved by any or 
a combination of the procedures discussed above. Also different codons for 
some or all of the codons used in the present DNA molecules could be 
substituted. These substituted codons may code for amino acids identical 
to those coded for by the codons replaced but result in higher yield of 
the polypeptide. Alternatively, the replacement of one or a combination of 
codons leading to amino acid replacement or to a longer or shorter carrier 
protein-like polypeptide may alter its properties in a useful way (e.g., 
increase the stability, increase the solubility, increase the therapeutic 
activity). 
Finally, the activity of the polypeptides produced by the recombinant DNA 
molecules of this invention may be improved by fragmenting, modifying or 
derivatizing the DNA molecules or polypeptides of this invention by 
well-known means, without departing from the scope of this invention. 
While we have described certain embodiments of the invention, it is 
apparent that those embodiments can be altered to provide other 
embodiments which utilize the processes and compositions of the invention. 
The scope of the invention is defined by the following claims rather than 
by the specific embodiments that have been presented by way of example.