t-PA mutant GK1L

Details t-PA mutant GK1L t-PA mutant GK1L

1991-04-16

1993-06-29

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435226, 435 696, 4352402, A61K 3748, C12N 950, C12N 964, C12N 1558

Patent

active

052234223

DESCRIPTION:

BRIEF SUMMARY
Human tissue plasminogen activator (t-PA) is a serine protease with a molecular weight of 68000 daltons which converts the pro-enzyme plasminogen into the active serine protease plasmin. Plasmin dissolves fibrin which is the main component of the protein matrix of coagulated blood. t-PA has a high affinity for fibrin and is also activated by fibrin (see Fibrinolysis 2 (1988), 133-142). t-PA is therefore of great medical interest.
An advantage of t-PA compared to other known plasminogen activators, such as e.g. urokinase or streptokinase, is the ability to stimulate its catalytic activity by fibrin (see J. Biol. Chem. 257 (1982), 2912-2919; Biochem. Biophys. Acta 755 (1983) 531-533).
t-PA (amino acid sequence cf. Vehar et al., Bio/Technology 2 (1984) 1051-1057) in its single-chain form consists of a heavy chain (H chain) and a light chain (L chain) which are held together by a disulphide bridge. The two-chain form is formed from a single-chain precursor form by specific cleavage with plasmin or other proteases between the amino acids (aa) 275 and 276. The heavy L chain of 32000 daltons weight contains the enzymatically active region which has homologies to other serine proteases such as urokinase or plasmin (Proc. Natl. Acad. Sci. USA 81 (1984), 5335-5339). The domains on the H chain of 39000 daltons weight are the finger domain (F) with homology to fibronectin (aa 1-49), the growth factor domain (G) with homology to mouse and human epidermal growth factor (aa 50-87) and two kringle domains, K1 (aa 88-175) and K2 (aa 176-262) with homology to the kringle structures in plasminogen.
Concerning the function of the individual domains of the H chain it is already known that only the domains K2 or/and F but not the domain K1 are responsible for binding of t-PA to fibrin and thus for the ability to stimulate the catalytic activity of t-PA in the presence of fibrin (EP-A-0 234 051).
It is known from EMBO 7 (1988) 2731-2740 that the activity of a t-PA mutant containing the complete domains K1 and F as well as a part of domain K2 of the H chain can also be stimulated by fibrin.
It is thus not clear to what extent the individual domains of the H chain effect the activity of the t-PA molecule with regard to fibrin and to what extent they cause a stimulation of the plasminogen cleaving activity.
The object of the present invention is to provide a t-PA mutant which has greater plasminogen cleaving activity than t-PA and whose catalytic activity can also be stimulated by fibrin or fibrinogen.
This object is achieved according to the present invention by the production of a recombinant DNA which codes for a protein with the domains GK1L of t-PA whereby the sequences coding for the domains K2 and F or sequences derived therefrom within the scope of the degeneration of the genetic code are completely deleted i.e. according to the exact exon/intron borders on the t-PA gene. The nucleotides 715 to 972 and 199 to 339 of the t-PA cDNA are therefore missing from the recombinant DNA according to the present invention (numbering according to Nature 301, (1983), 214-221). Surprisingly the ability to stimulate the catalytic activity by fibrin is preserved in GKIL although in the t-PA mutant GK1L the domains K2 and F of the H chain which are regarded as absolutely essential for stimulating the activity are completely missing from the gene product of the recombinant DNA according to the present invention. Surprisingly the catalytic activity of a supernatant from cells which express GK1L is even significantly higher than the activity of a supernatant from cells which express t-PA.
The invention also provides a process for the production of recombinant DNA according to the present invention from a DNA sequence coding for t-PA or for a t-PA mutant which contains more than the domains G, K1 and L by deletion of those sequences which do not code for the domains G, K1 and L while maintaining the exact exon/intron borders on the t-PA gene. A process is particularly preferred in which the deletion of the DNA coding for the domains K2 and F is

REFERENCES:
Kagitani et al., FEBS. 189(1): 145-149 (1985).
Stern et al., Gene 79: 333-344 (1989).
Stern et al., Gene 87: 305-308 (1990).

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