Heterologous production of streptokinase as a secretary form in Streptomyces lividans and nonsecretary form in Escherichia coli

Abstract

The skc gene encoding streptokinase (SK) with a molecular mass of approximately 47.4 kDa was cloned from Streptococcus equisimilis ATCC 9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the promoter for sprT [Streptomyces griseus trypsin (SGT)] was used in the host S. lividans TK24, a 47.4-kDa protein was detected along with a smaller hydrolyzed protein (44 kDa), suggesting that posttranslational hydrolysis had occurred as has been reported in other expression systems. The casein/plasminogen plate assay revealed that the plasmid construct containing the SGT signal peptide was superior to that containing the SK signal peptide in terms of SK production. Maximal production of SK was calculated to be about 0.25 unit/ml of culture broth, a value that was five times higher than that obtained with other expression systems using ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(∆DE3)pLys under the control of the T7 promoter, a relatively large amount of SK was expressed in soluble form without hydrolysis. SK activity in E. coli/pET28a-T7 p SK m was more than 2 units/ml of culture broth, even though about half of the expressed protein formed an inactive inclusion body.

The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks

“…Modifications of shake flasks by the introduction of baffles and other enhancements (like stainless steel spring coils) are frequently necessary in order to provide sufficient aeration and shear stress [ 25 , 29 , 30 ]. In S. lividans cultures, coiled and baffled flasks are normally used [ 30 - 33 ], but no detailed study has been done on how the geometry of the flasks can affect recombinant protein production, and its O -glycosylation as a function of the morphology of the filamentous bacteria.…”

Cloning, secretory expression, partial characterization, and structural modeling of an alkaline protease from Bacillus subtilis D-2

“…To optimize the secretory expression condition, different temperatures (12, 17, 22, 27, 32, and 37 °C), inducer concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol l -1 IPTG), and inducing times (0, 2, 4, 6, 8 and 10 h), were set to induce expression, followed by testing of the A660 and protease activity of the culture (Kim et al 2010).…”

Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

“…However, due to low SK production yields from natural host and its pathogenicity, so research interest has shifted to cloning and expression of SK in hyperproductive and safe heterologous host systems. Therefore, sk genes have been cloned and expressed in different expression systems including Bacillus subtilis [ 3 ], Streptococcus sanguis [ 4 ], Streptomyces lividans [ 5 , 6 ], Schizosaccharomyces pombe [ 7 ], Pichia pastoris [ 1 , 8 ], Lactococcus lactis [ 9 ], and Escherichia coli [ 10 , 11 ]. However, there are some disadvantages of producing recombinant proteins in Pichia pastoris due to high glycosylation level [ 12 ] or in Lactococcus lactis due to low cell density [ 9 ].…”