Preliminary Study On Directed Evolution Of Staphylokinase

Staphylokinase (Sak) is a 15.5kDa protein from some strains of Staphylococcus aureas that activate plasminogen by forming a 1:1 complex with plamin. Sak-plamin complex ,which has proteolytic activity , can form an enzyme-substrate complex with another plaminogen molecule, and efficiently convert the substrate plasminogen to an active plasmin. It is at least equipotent to rt-PA for coronary artery recanalization, significantly more fibrin-selective and obtainable in high yield by cytosolic expression in Escherichia coli. In addition, Sak has been shown to be more efficient than t-PA for the dissolution of platelet-enriched and retracted blood clots. Therefore in recent years, Sak become a promising thrombolytic reagent and stimulated much structural and protein engineering reseach.However, being a heterologous bacterial protein, its administration induces high titers of neutralizing antibodies formation in a majority of patients . In addition, Sak, like other plasminogen activators, has short half-life. We suppose enhancing activity of Sak is one way to solve these problems partly .Directed evolution is a new powerful approach for improving virtually any property of a therapeutic protein for which an appropriate screen can be devised in the absence of any knowledge of spatial structure and functional mechanism in advance. It mainly mimics natural' random mutagenesis and recombination in the laboratory. Wild-type Sak is modified by directed evolution for obtaining the mutants with enhanced enzyme activity and the optimized approach for Sak directed evolution is explored in this study.Error-prone PCR to introduce random mutations and screening of the resultant mutant libraries have been used to enhance the activity of wild-type Sak. The condition for error-prone PCR induced mutation was optimized in this study. The mutation rate was controlled to 1- to 2-amino-acid substitution in each gene by limiting the concentration of Mn2+ to 0.15mmol/L in error-prone PCR. Approximately 10000 active clones were identified by screening on an plasma agar plate. 100 potential high activity colonies were picked and screened by the method of fibrin agarplate pore-forming. Three variants with 1.5-fold and 2-fold activity increased over the wild-type parent were identified and sequenced .The three variants were used as a pool for the parents to initiate DNA shuffling. The mutant libraries were established and screened on plasma agar plates and approximately 10000 active clones were identified. 100 potential high activity clones were picked and screened by the method of fibrin agar plate pore-forming. Two recombinants were identified among ten clones that were sequenced.We used stagger extension process (StEP) to recombine the three varants that identified during error-PCR mutagenesis and screening. The recombined libraries were screened on plasma agar plates and approximately 10000 active clones were identified. 100 potential high activity clones were picked and screened by the method of fibrin agar plate pore-forming. Two recombinants were identified among ten clones that were sequenced. 100 potential high activity clones were picked and screened by the method of fibrin agar plate pore-forming. Two recombinants were identified among five clones that were sequenced.