Recombinant production of Streptococcus equisimilis streptokinase by Streptomyces lividans

Abstract

Background: Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.

Results: SK yield in the spent culture medium of S. lividans was higher when the Sec-dependent signal peptide mediates the SK translocation. Using a 1.5 L fermentor, the secretory production of the Vsi-SK fusion protein reached up to 15 mg SK/l. SK was partially purified from the culture supernatant by DEAE-Sephacel chromatography. A 44-kDa degradation product co-eluted with the 47-kDa mature SK. The first amino acid residues of the S. lividans-produced SK were identical with those of the expected N-terminal sequence. The Vsi signal peptide was thus correctly cleaved off and the N-terminus of mature Vsi-SK fusion protein released by S. lividans remained intact. This result also implicates that the processing of the recombinant SK secreted by Streptomyces probably occurred at its C-terminal end, as in its native host Streptococcus equisimilis. The specific activity of the partially purified Streptomyces-derived SK was determined at 2661 IU/mg protein.

Conclusion: Heterologous expression of Streptococcus equisimilis ATCC9542 skc-2 in Streptomyces lividans was successfully achieved. SK can be translocated via both the Sec and the Tat pathway in S. lividans, but yield was about 30 times higher when the SK was fused to the Sec-dependent Vsi signal peptide compared to the fusion with the Tat-dependent signal peptide of S. lividans xylanase C. Small-scale fermentation led to a fourfold improvement of secretory SK yield in S. lividans compared to lab-scale conditions. The partially purified SK showed biological activity. Streptomyces lividans was shown to be a valuable host for the production of a world-wide important, biopharmaceutical product in a bio-active form.

Figure 1

Immunodetection of SK in extracellular fractions. Proteins from culture supernatants were precipitation with a mixture of chloroform and methanol (1:3, v/v). In each lane, proteins according to 100 μl spent culture medium were loaded. Lane 1, S. lividans [pOW15] 40 h; lane 2, 60 ng SK standard; lane 3, S. lividans [pOVsiSK] 30 h; lane 4, S. lividans [pOVsiSK] 40 h; lane 5, S. lividans [pOXlnCSK] 30 h; lane 6, S. lividans [pOXlnCSK] 40 h.

Figure 2

Secretory yield of recombinant SK correlated with biomass of Streptomyces lividans [pOVsiSK] grown in lab-scale conditions. SK concentration was determined by means of ELISA. Growth was estimated by measuring biomass dry weight (mg/ml), standard errors were between 0.1 and 0.2.

Figure 3

Purification of extracellular SK from S. lividans culture supernatants upon small-scale fermentation. (A) 10% SDS-PAGE stained with Coomassie blue R-250, and (B) Immunoblotting analysis using a monoclonal anti-SK antibody. Lane 1, 25 μg of crude extract of S. lividans TK24 [pOW15]; lane 2, 25 μg of material precipitated with (NH₄)₂SO₄ of S. lividans TK24 [pOW15]; lane 3, 25 μg of crude extract of S. lividans TK24 [pOVsiSK]; lane 4, 25 μg of proteins precipitated with (NH₄)₂SO₄ of S. lividans TK24 [pOVsiSK]; lane 5, 25 μg of pooled anion exchange chromatography protein fractions with 58% purity; lane 6, 1 μg of SK standard; lane 7, Broad-range protein molecular weight markers.