Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reissue Patent
2000-03-28
2003-08-26
Saoud, Christine J. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S069400, C435S069800, C536S023400, C536S023510, C530S399000
Reissue Patent
active
RE038240
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates to recombinant DNA-directed synthesis of certain proteins. More particularly, this invention relates to endothelial cell growth factor (ECGF), its recombinant DNA-directed synthesis, and ECGF's use in the treatment of endothelial cell damage and/or regeneration.
(2) The Prior Art
Endothelial cell growth factor, referred to herein as “ECGF”, is a mitogen for endothelial cells in vitro. Growth of endothelial cells is a necessary step during the process of angiogenesis [Maciag, Prog. Hemostasis and Thromb., 7:167-182 (1984); Maciag, T., Hoover, G. A., and Weinstein, R., J. Biol. Chem., 257: 5333-5336 (1982)]. Bovine ECGF has been isolated by Maciag, et al., [Science 225:932-935 (
1984)] using streptomycin sulfate precipitation, gel exclusion chromatography, ammonium sulfate precipitation and heparin-
Sepharose
SEPHAROSE
affinity chromatography. Bovine ECGF purified in this manner yields a single-chain polypeptide which possesses an anionic iso-electric point and an apparent molecular weight of 20,000 [Maciag, supra; Schreiber, et al., J. Cell Biol., 101:1623-1626 (1985); and Schreiber, et al., Proc. Natl. Acad. Sci., 82:6138-6142 (1985)]. More recently, multiple forms of bovine ECGF have been isolated by Burgess, et al., [J. Biol. Chem. 260:11389-11392 (
1985)] by sodium chloride gradient elution of bovine ECGF from the heparin-
Sepharose
SEPHAROSE
column or by reversed-phase high pressure liquid chromatography (HPLC). The two isolated polypeptides, designated alpha-and beta-ECGF have apparent molecular weights of 17,000 and 20,000, respectively. Using this procedure, the bovine ECGF contained in 8,500 ml of bovine brain extract
(
6.25×107
(6.25×10
7
total units) is concentrated into a total of 6 ml of alpha-ECGF
(3.0×106
(
3
.
0
×
10
6
units) and 3 ml of beta-ECGF
(5.2×105
(
5
.
2
×
10
5
units). This is a 9,300-fold purification of alpha-ECGF and 16,300-fold purification of beta-ECGF (Burgess, supra.). Recently, murine monoclonal antibodies against bovine ECGF have been produced (Maciag, et al., supra.) which may be useful in purifying bovine ECGF in a manner similar to the monoclonal antibody purification of Factor VIIIC described by Zimmerman and Fulcher in U.S. Pat. No. 4,361,509.
In general, recombinant DNA techniques are known. See Methods In Enzymology, (Academic Press, New York) volumes 65 and 68 (1979); 100 and 101 (1983) and the references cited therein, all of which are incorporated herein by reference. An extensive technical discussion embodying most commonly used recombinant DNA methodologies can be found in Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982). Genes coding for various polypeptides may be cloned by incorporating a DNA fragment coding for the polypeptide in a recombinant DNA vehicle, e.g., bacterial or viral vectors, and transforming a suitable host. This host is typically an Escherichia coli (E. coli) strain, however, depending upon the desired product, eukaryotic hosts may be utilized. Clones incorporating the recombinant vectors are isolated and may be grown and used to produce the desired polypeptide on a large scale.
Several groups of workers have isolated mixtures of messenger RNA (mRNA) from eukaryotic cells and employed a series of enzymatic reactions to synthesize
doublestranded
double-stranded
DNA copies which are complementary to this mRNA mixture. In the first reaction, mRNA is transcribed into a singlestranded complementary DNA
(cDNA)
(ss-cDNA)
by an RNA-directed DNA polymerase, also called reverse transcriptase. Reverse transcriptase synthesizes DNA in the 5′-3′ direction, utilizes deoxyribonucleoside 5′-triphosphates as precursors, and requires both a template and a primer strand, the latter of which must have a free 3′-hydroxyl terminus. Reverse transcriptase products, whether partial or complete copies of the mRNA template, often possess short, partially double-stranded hairpins (“loops”) at their 3′ termini. In the second reaction, these “hairpin loops” can be exploited as primers for DNA polymerases. Preformed DNA is required both as a template and as a primer in the action of DNA polymerase. The DNA polymerase requires the presence of a DNA strand having a free 3′-hydroxyl group, to which new nucleotides are added to extend the chain in the 5′-3′ direction. The products of such sequential reverse transcriptase and DNA polymerase reactions still possess a loop at one end. The apex of the loop or “fold-point” of the double-stranded DNA, which has thus been created, is substantially a single-strand segment. In the third reaction, this single-strand segment is cleaved with the single-strand specific nuclease S1 to generate a “blunt-end” duplex DNA segment. This general method is applicable to any mRNA mixture, and is described by Buell, et al., J. Biol. Chem., 253:2483 (1978).
The resulting double-stranded cDNA mixture (ds-cDNA) is inserted into cloning vehicles by any one of many known techniques, depending at least in part on the particular vehicle used. Various insertion methods are discussed in considerable detail; in Methods In Enzymology, 68:16-18 (1980), and the references cited therein.
Once the DNA segments are inserted, the cloning vehicle is used to transform a suitable host. These cloning vehicles usually impart an antibiotic resistance trait
on
to
the host. Such hosts are generally prokaryotic cells. At this point, only a few of the transformed or transfected hosts contain the desired cDNA The sum of all transformed or transfected hosts constitutes a gene “library”. The overall ds-cDNA library created by this method provides a representative sample of the coding information present in the mRNA mixture used as the starting material.
If an appropriate oligonucleotide sequence is available, it can be used to identify clones of interest in the following manner. Individual transformed or transfected cells are grown as colonies on a nitrocellulose filter paper. These colonies are lysed; the DNA released is bound tightly to the filter paper by heating. The filter paper is then incubated with a labeled oligonucleotide probe which is complementary to the structural gene of interest. The probe hybridizes with the cDNA for which it is complementary, and is identified by autoradiography. The corresponding clones are characterized in order to identify one or a combination of clones which contain all of the structural information for the desired protein. The nucleic acid sequence coding for the protein of interest is isolated and reinserted into an expression vector. The expression vector brings the cloned gene under the regulatory control of specific prokaryotic of eukaryotic control elements which allow the efficient expression (transcription and translation) of the ds-cDNA. Thus, this general technique is only applicable to those proteins for which at least a portion of their amino acid or DNA sequence is known for which an oligonucleotide probe is available. See, generally, Maniatis, et al., supra.
More recently, methods have been developed to identify specific clones by probing bacterial colonies or phage plaques with antibodies specific for the encoded protein of interest. This method can only be used with “expression vector” cloning vehicles since elaboration of the protein product is required. The structural gene is inserted into the vector adjacent to regulatory gene sequences that control expression of the protein. The cells are lysed, either by chemical methods or by a function supplied by the host cell or vector, and the protein is detected by a specific antibody and a detection system such as enzyme immunoassay. An example of this is the lambda
gt
11
gt
11
system described by Young and Davis, Proc. Nat'l. Acad. Sci. USA, 80:1194-1198 (1983) and Young and Davis, Science 222:778 (1983).
SUMMARY OF THE INVENTION
The present invention has made it possible to provid
Burgess Wilson
Drohan William N.
Jaye Michael C.
Maciag Thomas
Aventis Pharmaceuticals Inc.
Saoud Christine J.
Turner Sharon
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