277312 Strategies for Enhanced Production of Recombinant Staphylokinase by Escherichia Coli

Tuesday, October 30, 2012: 9:24 AM
Westmoreland East (Westin )
Arshad Jawed, K.L. Dikshit and Debendra K. Sahoo, CSIR - Institute of Microbial Technology, Chandigarh, India

Thromboembolism, a significant medical problem, is the formation of clot in blood vessel and this disorder has emerged as one main cause of mortality around the globe. Plasmin, the active fibrinolytic component of circulating system, dissolves clots through limited proteolysis. The 15.5 kDa bacterial protein staphylokinase (SAK) isolated from Staphylococcus aureus, a non-enzymatic, forms a stochiometric protein-protein complex with plasmin(ogen). Cleavage of the peptide bond Lys10-Lys11 of SAK in this complex is the trigger for the formation of an enzyme species capable of activating plasminogen. The SAK-plasmin(ogen) complex shows a high degree of specificity for cleavage of blood clots with reduced side effects as the SAK-plasmin(ogen) complex is strongly inhibited by  α2-antiplasmin circulating in the blood. Owing to the market potential, we aimed for development of a large-scale process for production of staphylokinase at reduced cost.

The application of recombinant DNA technology to large-scale processes has enabled the production of therapeutic proteins in quantities that might otherwise have been difficult, if not impossible, to obtain from natural sources. The extensive employment in recombinant protein production and vast knowledge base on genetics and physiology of gram-negative Escherichia coli along with its ease of handling, inexpensive growth requirements and the accumulation of the product to higher levels in the cell cytoplasm has made it to be called as the “microbial factory”. Improvements in fermentation processes is known to be an important avenue for increasing yield of a recombinant protein in E. coli and the efficiency of recombinant protein expression in Escherichia coli depends on the parameters affecting both cell growth and protein expression. The preferred method for increasing the volumetric yield of heterologous recombinant proteins, which is proportional to both cell density (unit cell mass per litre) and specific yield (amount of product per unit cell mass), is the fed-batch strategy.

We formulated strategies for large-scale production of staphylokinase, using E. coli cells transformed with plasmid pET-9b containing the gene for staphylokinase. The protein was expressed intracellularly in soluble form. Fed-batch cultivations were carried out that involved initial cell cultivation in batch mode followed by subsequent addition of nutrients using different feeding strategies to build up high density of E. coli cells for induction. We strategized to increase volumetric yield and productivity of target protein without compromising on its specific yield, i.e., protein yield per unit cell mass (SAK expression at shake flask level with LB medium was taken as control), thereby reducing the load of intracellular contaminants per unit of SAK produced at fermentation stage that was perceived to reduce purification cost and result in low processing cost of the target protein. Optimization of feeding was carried out to increase cell concentrations and SAK yield.  The effects of media constituents such as sources of carbon and organic nitrogen, C/N ratio, inducer concentration and process parameters such as dissolved oxygen concentration (DOC) on cell growth and SAK production were investigated. A medium mainly consisting of sugar (glucose), simple salts and some low cost organic nitrogen sources was developed to produce staphylokinase at a concentration of 3500 mg/L, highest reported up to date providing added advantages of processing lower quantity of broth per unit of staphylokinase. Staphylokinase so obtained exhibited biological properties e.g., clot dissolution. The process for production of Staphylokinase was scaled up to 15 L fermenter. The results in detail will be presented.

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