| abstract |
Human tissue plasminogen activator (t-PA) mutant (I) is produced by recombinant cells and is resistant to specific enzymatic cleavage. (I) is esp. in single-chain form, stabilised by site-directed mutagenesis at a 2-chain cleavage site. Partic. the matural Arg at position 275 is replaced by Gly or Glu, or Ila at position 276 is replaced. Also new are (1) DNA sequences coding for (I); (2) expression vectors able to express (I) and (3) microorganisms and cell cultures transformed with these vectors. A native t-PA gene, contg. the sequence to be mutated, is inserted into the phage M13mp8, and the single-strand form of the prod. annealed to a synthetic oligonucleotide, co-ng. 1 or more triplets coding for the amino acid(s) to be substd. forming a double-strand region. Expression vectors contg. the new DNA sequences can be expressed in E.coli or partic. cultures of Chinese hamster ovary (using vectors which include the SV40 origin of replication as promoter). - |