In Vitro Characterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt Inducible E. coli GJ1158
Figure 1
Cloning of “SAK- RGD- Hirulog” (SRH) and SAK gene into pRSET-A. (a) Amplification of mature SAK and synthetic gene, lane 1: amplification of mature SAK of 420 bp; lane 2 : 100 bp DNA ladder; lane 3: Mutual amplification of synthetic gene of 123 bp. (b) Restriction digestion analyses of SRH, mature SAK and RGD-Hirulog, Lane 1: Double digestion of recombinant plasmid pRSET-A-SRH. The SRH gene of size 543 bp was pop out; Lane 2: Double digestion of recombinant plasmid pRSET-A-SAK. The SAK gene of size 420 bp was pop out; Lane 3: Double digestion of recombinant plasmid pRSET-A-RGD-Hirulog. Synthetc gene of 123 bp was pop out; Lane 4: Triple digestion of recombinant plasmid pRSET-A-SRH. The SAK (420 bp) and synthetic gene “RGD-Hirulog (123 bp)” were pop out; Lane 5: Synthetic gene “RGD-Hirulog” of 123 bp; Lane 6: 100 bp DNA ladder. (c) PCR confirmation of pRSET-A-SAK. Lane 1: 100 bp DNA ladder; Lane 2: Amplification of mature SAK with gene specific primers; Lane 3: Amplification of mSAK with vector specific forward primers T7U and gene specific reverse primers; Lane 4: amplification of mature SAK with gene specific forward primers and vector specific reverse primes T7T. (d) PCR confirmation of pRSET-A-RGD-Hirulog. Lane 1: Mutual amplification of synthetic gene RGD-Hirulog; Lane 2: 100 bp DNA ladder; Lane 3: Amplification of RGD-Hirulog gene with vector specific forward primers T7U and gene specific reverse primers; Lane 4: Amplification of RGD-Hirulog gene with gene specific forward primer and vector specific reverse primer T7.T; Lane 5: Amplification of RGD-Hirulog gene with vector specific primers T7U and T7U.
