Primary structure and gene structure of staphylokinase

Abstract

The nucleotide sequence was determined of the 2.9 kb HindIII restriction fragment, obtained from the genomic DNA of a selected Staphylococcus aureus strain (strain no. 23) which directs high level expression of staphylokinase (STA) in E. coli JM83 cells transformed with the recombinant plasmid pUCSTAHH, consisting of pUC19 containing this 2.9 kb insert. The fragment contained an open reading frame of 489 base pairs (base pairs 996–1484) encoding 163 amino acids, with amino acids 28, 34 and 38 corresponding to the NH 2-terminal residue of the three variants (STA-M, STA-Δ6 and STA-Δ10) of recombinant STA (STAR) recovered from culture broth conditioned by transformed E. coli (Collen et al, Fibrinolysis, 1992; 6: 203–213). This coding sequence is preceded upstream by canonical Shine-Dalgarno, -10 and -35 prokaryotic promoter sequences and in addition by an open reading frame spanning nucleotides 50–802 which encodes an unknown protein of 251 amino acids. The sequence encoding STA is very similar to that previously cloned from bacteriophages Sø-c and 42D. Recombinant plasmids with a l.7kb AccI- EcoRI fragment insert (base pairs 683–2168) containing only the sequence encoding STA, directed excretion of STA activity to an extent comparable to that obtained with the recombinant plasmid containing the 2.9 kb HindIII restriction fragment insert.