Cloning and expression in Escherichia coli, Bacillus subtilis, and Streptococcus sanguis of a gene for staphylokinase — a bacterial plasminogen activator

Abstract

The gene coding for the bacterial plasminogen activator staphylokinase was cloned from the Staphylococcus aureus phage 42D, a serogroup F phage used for lysotyping, onto the standard Escherichia coli plasmid vector pACYC184. The coding and flanking sequences of the sak42D gene were largely identical to those of a sak gene cloned from the serologically different S. aureus phage SøC (Sako and Tsuchida 1983). Subcloning of a 2.5 kb phage 42D DNA fragment onto plasmid pGB3631 allowed the sak42D gene to be introduced into the gram-positive hosts Bacillus subtilis and Streptococcus sanguis. The sak42D gene was expressed and secreted most efficiently by B. subtilis cells (25 micrograms/ml of culture supernatant) reduced in exoprotease production. In this host expression and secretion of Sak was initiated at the early growth phase and continued through the logarithmic phase. Formation of Sak was, however, also observed with the other cloning hosts. The Sak elaborated by the heterologous hosts was serologically identical with authentic Sak derived from S. aureus.

Novel preparation protocol for the expression and purification of recombinant staphylokinase

“…The N-terminal amino acid sequence of the purified r-Sak was SSSFDKGKYKKGDDA- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). This result was the same as deduced from the DNA sequence.…”

“…Many clinical trials support the immense therapeutic potential of r-Sak for acute myocardial infarction [4,5], peripheral arterial occlusion [6,7] and pulmonary embolism [8][9][10]. The gene encoding Sak has been widely studied, and the rSAK protein has been produced in Escherichia coli, Bacillus subtilis and Streptomyces lividans [11][12][13][14][15][16][17][18]. r-Sak exists in a soluble state within the bacterium or in fermentation solutions.…”

Staphylokinase-Specific Cell-Mediated Immunity in Humans

“…Its fibrinolytic potency has already been recognized half a century ago (1). The staphylokinase gene has been cloned, among others, from a bacteriophage (Sak42D) and from a lysogenic S. aureus DNA (SakSTAR) (2,3). The encoded proteins, Sak42D and SakSTAR, have revealed a unique fibrin specificity (for review, see Ref.…”

Enhancement of Secretion and Extracellular Stability of Staphylokinase in Bacillus subtilis by wprA Gene Disruption

“…Thus, several efforts to produce SAK in large quantities have been made (2,13,21,29). A Bacillus expression system exploiting the natural promoter and translation signals of SAK resulted in the secretion of 25 mg of recombinant SAK (rSAK) per liter in B. subtilis DB104, which is deficient in two major extracellular proteases (2,7).…”