Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe.
Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus. Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with the mature streptokinase from Streptococcus sp. was constructed and inserted into the expression vector containing the thiamine-regulated promoter. We obtained a high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction. The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz. pombe.
Kumar
Raj
R
Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
Singh
Jagmohan
J
D000595:Amino Acid Sequence; D005656:Fungal Proteins; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics; D019897:Periplasm / Q000378:metabolism; D010675:Pheromones / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology; D013300:Streptokinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
D005656:Fungal Proteins; C093953:NMT1 protein, S pombe; D010675:Pheromones; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C085847:map2 protein, S pombe; D010447:Peptide Hydrolases; D013300:Streptokinase