The present invention is directed to dna encoding human endothelial cell growth factors, and to plasmids comprising said dna. In particular, the invention relates to dna encoding a cleavable signal peptide and an endothelial cell growth factor, wherein removal of said signal peptide yields a mature form of the growth factor.
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12. A plasmid comprising a dna encoding a cleavable heterologous signal peptide and human α-endothelial cell growth factor comprising having the amino acid sequence
n Y k K k L L Y C S n G G H f L R I L P DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK KNGSCKRGRRTHYGQKAILFLPLPVSSD, wherein removal of said signal peptide yields a mature form of said human α-endothelial cell growth factor.
13. A plasmid comprising a dna encoding a cleavable heterologous signal peptide and human β-endothelial cell growth factor comprising the amino acid sequence
AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYIS KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD, wherein removal of said signal peptide yields a mature form of said human β-endothelial cell growth factor.
5. A dna encoding a cleavable heterologous signal peptide and an endothelial cell growth factor, wherein removal of said signal peptide yields a mature form of said endothelial cell growth factor, and said endothelial cell growth factor either has the amino acid sequence of α-endothelial cell growth factor
(NYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHI QLQLSAEVSVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTY I S k K H A e k n w f V G L k KNGSCKRGPRTHYCQKAILFLPLPVSSD) or comprises the amino acid sequence of β-endothelial cell growth factor (AEGEITTFTALTAKFNLPPGNYKKPKLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYIS KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD).
1. An isolated dna encoding a cleavable signal peptide and an endothelial cell growth factor, wherein removal of said signal peptide yields a mature form of said endothelial cell growth factor, and said endothelial cell growth factor either has the amino acid sequence of α-endothelial cell growth factor
(NYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTYI S k K H A e k n w f V G L k KNGSCKRGPRTHYGQKAILFLPLPVSSD) or comprises the amino acid sequence of α-endothelial β-βendothelial cell growth factor (AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLYYGSQTPNEECLFLERLEENHYNTYIS KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD).
8. A plasmid comprising a dna encoding a cleavable signal peptide and an endothelial cell growth factor, wherein removal of said signal peptide yields a mature form of said endothelial cell growth factor, and said endothelial cell growth factor either has the amino acid sequence of α-endothelial cell growth factor
(NYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTY I S k K H A e k n w f V G L k KNGSCKRGPRTHYGQKAILFLPLPVSSD) or comprises the amino acid sequence of β-endothelial cell growth factor
(AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYIS KKHAQEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD).
2. An isolated dna according to
n Y k K P k L L Y C S n G G H f L R I L P DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMNTDGLLYGSQ TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK KNGSCKRGPRTHYGQKAILFLPLPVSSD.
3. An isolated dna according to
A e G e I T T f T A L T e k f n L P P G n Y k K P k L LYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQT P n e E C L f L e R L e E n H Y n T Y I S KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD.
4. An isolated dna according to
6. A dna according to
n Y k K P k L L Y C S n G G H f L R I L P DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTFDGLLYGSQ TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK KNGSCKRGPRTHYGQKAILFLPLPVSSD.
7. A dna according to
AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQT P n e E C L f L e R L e E n H Y n T Y I S KKHAEKNWFVGLKKNGSCKRGPRRTHYGQKAILFLPL PVSSD.
9. A plasmid according to
n Y k K P k L L Y C S n G G H f L R I L P DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTDGLYYGSQ TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK KNGSCKRGPRTHYGQKAILFLPLPVSSD.
10. A plasmid according to
A e G e I T T f T A L T e k f n L P P G n Y k K P k LLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQT P n e E C L f L e R L e E n H Y n T Y I S KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD.
0. 14. A process for expressing an endothelial cell growth factor in a host cell, comprising introducing the plasmid according to
0. 15. A process for expressing a human α-endothelial cell growth factor in a host cell, comprising introducing the plasmid according to
0. 16. A process for expressing a human β-endothelial cell growth factor in a host cell, comprising introducing the plasmid according to
0. 17. A process for expressing an endothelial cell growth factor in a host cell, comprising introducing the plasmid according to
0. 18. A process for expressing a human α-endothelial cell growth factor in a host cell, comprising introducing the plasmid according to
0. 19. A process for expressing a human β-endothelial cell growth factor in a host cell, comprising introducing the plasmid according to
0. 20. The process according to
0. 21. The process according to
0. 22. A process for preparing an endothelial cell growth factor, comprising transforming a host cell with a plasmid according to
0. 23. A process for preparing a human α-endothelial cell growth factor, comprising transforming a host cell with a plasmid according to
0. 24. A process for preparing a human β-endothelial cell growth factor, comprising transforming a host cell with a plasmid according to
0. 25. A process for preparing an endothelial cell growth factor, comprising transforming a host cell with a plasmid according to
0. 26. A process for preparing a human α-endothelial cell growth factor, comprising transforming a host cell with a plasmid according to
0. 27. A process for preparing a human β-endothelial cell growth factor, comprising transforming a host cell with a plasmid according to
0. 28. The process according to
0. 29. The process according to
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This is a continuation of application Ser. No. 08/743,261 filed on Nov. 4, 1996, gt11
A. Introduction
As used herein, "ECGF" denotes endothelial cell growth factor or its fragments produced by cell or cell-free culture systems, in bioactive forms having the capacity to influence cellular growth, differentiation, and migration in vitro as does ECGF native to the human angiogenic process.
Different alleles of ECGF may exist in nature. These variations may be characterized by differences in the nucleotide sequence of the structural gene coding for proteins of identical biological function. It is possible to produce analogs having single or multiple amino acid substitutions, deletions, additions, or replacements. All such allelic variations, modifications, and analogs resulting in derivatives of ECGF which retain the biologically active properties of native ECGF are included within the scope of this invention.
The glycosaminoglycan heparin potentiates the mitogenic effect of both bovine and recombinant human ECGF. Heparin naturally exists as a heterogeneous mixture of polysaccharide chains ranging from about 6,000 to about 25,000 Da (Alberts et al. in "Molecular Biology of the Cell" Garland Publishing, Inc. (1983) pp. 692-715). Low molecular weight heparins (LMWH) having a variety of advantages over natural heparin have been prepared (see U.S. Pat. No. 4,401,662; 4,446,314; 4,826,827; 5,032,679 and Mestre et al. Thrombosis Research 38, 389-399 (1985)) and are also useful in the practice of the present invention.
"Expression vectors" refer to vectors which are capable of transcribing and translating DNA sequences contained therein, where such sequences are linked to other regulatory sequences capable of affecting their expression. These expression vectors must be replicable in the host organisms or systems either as episomes, bacteriophage, or as an integral part of the chromosomal DNA. One form of expression vector which is particularly suitable for use in the invention is the bacteriophage, viruses which normally inhabit and replicate in bacteria. Particularly desirable phage for this purpose are the lambda gt10 and gt11 phage described by Young and Davis, supra. Lambda 53366 53336
Jaye, Michael C., Burgess, Wilson, Maciag, Thomas, Drohan, William N.
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