Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system

Wong, Sui‐Lam; Ye, Ruiqiong; Nathoo, S.

doi:10.1128/aem.60.2.517-523.1994

Cited by 54 publications

(25 citation statements)

References 31 publications

(22 reference statements)

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“…Protease-deficient Bacillus strains, such as B. subtilis derivative WB600, have thence been genetically engineered to circumvent host-mediated proteolysis ( Figure 1F and Table 1) [56], though it only remained a partial success due to the persisting proteolytic activity [57]. Nonetheless, WB600 yielded higher productivity of recombinant proteins including β-lactamase [56], streptokinases [89], and several others. Subsequent removal of the remaining protease-encoding genes vpr and wprA in WB600 has led to an increased stability of the expressed protein molecules [90].…”

Section: Bacillus Subtilis As a Versatile Host With Highly Efficient mentioning

confidence: 99%

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

Kim

Choe

Lee

et al. 2020

IJMS

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A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

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Section: Bacillus Subtilis As a Versatile Host With Highly Efficient mentioning

confidence: 99%

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

Kim

Choe

Lee

et al. 2020

IJMS

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“…Site-directed mutagenesis of the streptokinase production plasmid. Plasmid pSK3 (48) is an expression vector in B. subtilis that is used to produce streptokinase with the sucrose-inducible regulatory region from B. subtilis sacB, encoding levansucrase, to control the expression. In this expression vector, secretion of streptokinase is directed by the sacB signal sequence (41,47).…”

Section: Methodsmentioning

confidence: 99%

“…To determine the significance of the Nterminal processing event and to explore the possibility of developing engineered forms of streptokinase with longer functional half-lives, we report the development of various forms of streptokinase through site-directed mutagenesis. These mutant proteins (abbreviated as muteins) of streptokinase were produced by the Bacillus subtilis secretory production system (48). Biochemical characterization and in vitro processing studies of these purified streptokinase muteins illustrate that plasmin-resistant streptokinase can be developed.…”

mentioning

confidence: 99%

Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis : Streptokinase with Longer Functional Half-Life

Duan

et al. 1998

Appl Environ Microbiol

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The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilisWB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.

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“…The H46A is also the main source of the SK gene that has been expressed in various other microorganisms. 134 After the successful sequencing of SK gene (skc) from S. equisimilis H46Ascientists became able to use this gene in producing SK safely in nonpathogenic bacteria. It has been expressed in B. subtilis WB600, E. coli and the yeast Pichia pastoris.…”

Section: Streptokinasementioning

confidence: 99%

The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi

Kotb

2014

Biotechnology Progress

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Formation of endogenous thrombi in blood vessels is one of the leading causes of death in our modern life. According to data provided by the World Health Organization (WHO) in 2000, heart diseases are responsible for 29% of the total mortality rate in the world. For this, a tremendous amount of research has been done in the area of prevention and treatment of these diseases. The classical therapy of these thrombi relies upon the use of antiplatelets, anticoagulants, or even surgeries. Relatively recently, the fibrinolytic enzymes produced by microorganisms, snakes, earthworms, insects, plants, and other organisms are being successfully used in the treatment of blood clots, especially with regard to the direct dissolving action on fibrin in tandem with less cost and side effects in comparison with the first-generation thrombolytic agents, streptokinase and urokinase. Furthermore, recombinant DNA technology has succeeded in improving and decreasing the undesirable effects of the first generation of enzymes. Recombinant PAs or rt-PAs like alteplase, retelase, saruplase, tenecteplase, lanoteplase, and desmoteplase became available in the drug markets with advantages of less binding loci with PAI-1 to avoid degradation while providing faster and more complete reperfusion in a greater number of patients with less risk of bleeding and intracranial hemorrhage. This review is the first to cover all the natural and recombinant thrombolytic agents used in enzyme therapy.

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Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system

Cited by 54 publications

References 31 publications

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis : Streptokinase with Longer Functional Half-Life

The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi

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