Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Abstract

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.

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.…”

Glycosylation of Asparagine-28 of Recombinant Staphylokinase with High-Mannose-type Oligosaccharides Results in a Protein with Highly Attenuated Plasminogen Activator Activity

“…14), the Sak-hPg complex must be processed by another hPg activator to provide Sak-hPm, which then functions as a hPg activator complex. During the conversion of hPg to hPm by Sak, a decapeptide is cleaved from the amino terminus of Sak (1). Although this peptide cleavage is not a rate-limiting step in hPg activation (15), it has been concluded that this event is necessary for this activation to occur (16).…”

Immediate entrance to the export pathway after synthesis as a requirement for export of the sak gene product in Escherichia coli

“…There have been reported, at the same time, several examples in which heterospecific secreted proteins are exported into the periplasmic space in E. coli (15,28,29, 32). However, the molecular nature of these phenomena remains to be solved.Recently, we have cloned the structural gene for staphylokinase (SAK), which is an extracellular protein produced by certain strains of Staphylococcus aureus, onto plasmid vector pBR322 and have shown that functional SAK is produced and exported into the periplasmic space of E. coli cells (24). From the amino acid sequence of the gene product deduced from the nucleotide sequence (25) and SAK purified from E. coli cells (23), we have identified the signal peptide of 27 amino acids.…”

“…Recently, we have cloned the structural gene for staphylokinase (SAK), which is an extracellular protein produced by certain strains of Staphylococcus aureus, onto plasmid vector pBR322 and have shown that functional SAK is produced and exported into the periplasmic space of E. coli cells (24). From the amino acid sequence of the gene product deduced from the nucleotide sequence (25) and SAK purified from E. coli cells (23), we have identified the signal peptide of 27 amino acids.…”