Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-08-15
2003-11-04
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S069500, C435S071200, C435S003000, C435S005000, C435S006120, C435S007370, C435S235100, C435S325000
Reexamination Certificate
active
06642026
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The field of the invention relates to methods of producing a recombinant fibroblast growth factor protein and its use in promoting angiogenesis.
2. Description of the Related Art
Fibroblast growth factors (FGF) are nine structurally related polypeptides, which are potent regulators of cell proliferation, differentiation and normal development. They also take part in pathological processes of tumorogenesis and metastasis (Galzie, et al. Biochem. Cell Biol. (1997) 75: 669-685). They are potent mitogens and differentiation factors for a broad range of mesoderm and neuroectoderm derived cells, including endothelial cells.
The heparin proteoglycans, heparin or heparin sulfate, bind several FGF molecules together as a complex which are presented to the FGF receptors. FGF proteins bind to their receptors resulting in the activation of protein tyrosine kinases. The phosphorylation of these tyrosine kinases initiates multiple signals including the transcription of new mRNA's.
Two fibroblast growth factors, basic and acidic, are described as potent inducers of angiogenesis (Friesel et al. (1995) FASEB J. 9: 919-925). Both basic and acidic factors have been implicated in the control of blood vessel formation and their involvement in normal and pathological angiogenesis (Slavin, J. (1995) Cell Biology International 19(5): 431-444). These factors have been purified, their amino acid sequences have been determined and their cDNA has been cloned and sequenced.
Acidic Fibroblast Growth Factor (aFGF) has been described under various names including embryonic kidney-derived angiogenesis factor I, astroglial growth factor I, endothelial cell growth factor (ECGF), retina-derived growth factor, heparin-binding growth factor class 1, endothelial growth factor, eye-derived growth factor II, prostatropin, and glial maturation factor (Gospodarowicz, et al. (1987) Journal of Cellular Physiology supplement 5: 15-26). Cloning, nucleotide sequence and chromosome localization have been described (Jaye et al. (1986) Science 233: 541-545).
The aFGF gene is situated on chromosome 5. It has a single copy and encodes three exons separated by two introns. A 4.8 kb mRNA translates synthesis of a form of aFGF with 155 amino acids. However, the N-terminal methionine residue is removed in vivo to give a 154 amino acid form. This 154 amino acid form of the aFGF is processed into two forms which are 140 and 134 amino acids. The aFGF protein is an anionic mitogen of molecular weight 15,000-17,000 D.
The aFGF protein has been found in brain, retina, bone matrix and osteosarcoma. Only forms with 140 and 134 amino acids have been obtained from tissues. It has been suggested that the truncated aFGF forms are an artifact created by specific proteases during aFGF extraction and isolation (Gospodarowicz, et al. (1987) Journal of Cellular Physiology supplement 5: 15-26; Jaye et al. (1987) The Journal of Biological Chemistry 262 (34):16612-16617).
It has been suggested that heparin potentiates the biological activity of the aFGF protein (Thornton et al. (1983) Science 222 (4624): 623-625). Heparin binding to aFGF has been observed (Maciag et al. (1984) Science 225 (4665): 932-935). This heparin-binding characteristic has been used as an efficient affinity chromatography method for the purification of aFGF protein. Heparin potentiates the biological activity of aFGF and the enhanced activity of the aFGF-heparin complex varies from several to one hundred fold (Lobb, et al. (1986) Anal. Biochem. 154: 1-14).
SUMMARY OF THE INVENTION
In one embodiment, the present invention relates to a method for producing a biologically active human acidic fibroblast growth factor protein, including the steps of:
transforming a strain of
E. coli
with a plasmid having at least one copy of an expressible gene encoding a biologically active human acidic fibroblast growth factor protein, operably linked to a promoter;
infecting the transformed bacterial host cell with a bacteriophage &lgr; capable of mediating delayed lysis; and
cultivating the
E. coli
host cell under a culture condition that induces lytic growth of said cell without lysis until a desired level of production of said protein is reached, wherein said protein is produced as a soluble, biologically-active human acidic fibroblast growth factor protein.
In a preferred embodiment, the bacteriophage &lgr; has a temperature-sensitive mutation. In a more preferred embodiment, the temperature-sensitive mutation is cI
857
.
In a preferred embodiment, the
E. coli
host cells are grown at a temperature which prevents lytic growth of the bacteriophage &lgr; prior to the cultivating step.
In a preferred embodiment, the bacteriophage &lgr; has a mutation in at least one gene capable of mediating delayed lysis. In a more preferred embodiment the at least one gene capable of mediating delayed lysis is selected from the group consisting of N, Q and R.
In a preferred embodiment, the strain of
E. coli
produces a suppressor for the repair of amber-mutations.
In an alternate embodiment, the strain of
E. coli
lacks a suppressor for the repair of amber-mutations.
In a preferred embodiment, the infecting bacteriophage &lgr; is provided at a multiplicity of infection in a range of about 1 to about 100. In a more preferred embodiment, the infecting bacteriophage &lgr; is provided at a multiplicity of infection in a range of about 10 to about 25.
In a preferred embodiment, the bacteriophage-mediated delayed lysis of the strain of
E. coli
is delayed at higher multiplicities of infection relative to lower multiplicities of infection.
In a preferred embodiment, the biologically active human acidic fibroblast growth factor protein contains 154 amino acids. In a more preferred embodiment, the human acidic fibroblast growth factor protein has the sequence as set forth in SEQ ID NO: 8.
In a preferred embodiment, the promoter is a T7 polymerase promoter and the
E. coli
strain is capable of expressing the gene for T7 RNA polymerase. In a more, preferred embodiment, the gene for T7 RNA polymerase gene is under the control of an inducible promoter. In an even more preferred embodiment, the inducible promoter is a lac UV 5 promoter.
In an alternate embodiment, the biologically active human acidic fibroblast growth factor protein contains 146 amino acids.
In another embodiment, the biologically active human acidic fibroblast growth factor protein contains 140 amino acids.
In another embodiment of the invention, the biologically active human acidic fibroblast growth factor protein contains 134 amino acids.
In a preferred embodiment, a method of producing a biologically active human acidic fibroblast growth factor protein is provided which comprises:
a) growing a first strain of
E. coli
cells, which harbor a strain of bacteriophage &lgr;, wherein the bacteriophage &lgr; has a temperature-sensitive mutation,
b) adjusting the temperature to provide for lysis of the first strain of
E. coli
cells and release of the bacteriophage &lgr;,
c) providing a second strain of
E. coli
cells which have been transformed with a plasmid having at least one copy of an expressible gene encoding said biologically active human acidic fibroblast growth factor protein, said expressible gene being operably linked to a T7 polymerase promoter under the control of an inducible promoter, wherein the second strain of
E. coli
cells may be induced to express the gene for T7 RNA polymerase by addition of an inducer;
d) infecting the second strain of
E.coli
cells with the bacteriophage &lgr; released from the first strain of
E. coli
cells; and
e) incubating the infected second strain of
E. coli
cells in a culture medium containing the inducer, such that protein is produced and released into the culture medium upon lysis of the second strain of
E. coli
cells, wherein said protein is produced as a soluble, biologically-active protein at a concentration greater than 100 microgram/ml.
Another aspect of the invention encompasses a chemically synthesized nucleic acid having the se
Chernykh Svitlana I.
Kordyum Vitaliy A.
Slavchenko Iryna Yu.
Stegmann Thomas J.
Vozianov Oleksandr F.
Housel James
Knobbe Martens Olson & Bear LLP
Li Bao Qun
Phage Biotechnology Corporation
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