Protein engineering of human tissue type plasminogen activator (tPA) using active site of plant-cuscutain enzyme to increase its permanent and activity

Abstract

tPA is a fibrinolitic agent that cause dissolves of blood clots and used in stroke and cardiovascular diseases. Cuscutain is a stable and highly active protease in plants such as dodder, and parasite plants using this enzyme to penetrate into host. Although produced cost of recombinant proteins in plants is 10-50 times less than bacteria, but production of recombinant proteins in plants has problems such as difficulty of downstream process, low stability and activity of purified recombinant proteins. To increase stability and activity of tPA using protein engineering, dodder-cuscutain active site was spliced to tPA. DNA was extracted from dodder plants and desired segments of cuscutain amplified using specific primers. Then, the segment of cuscutain was shuffled to tPA by SOEing PCR method. Constructed chimeric tPA was cloned under control of CaMV 35S promoter and NOS terminator. Kozak enhancer sequence and signal peptide sequence (KDEL) were added to amino and carbocyclic terminus, respectively. Construct of pBI(tPA) was transferred to tobacco plants using agrobacterium method and transgenic plants selected on kanamycin medium. Analysis of transgenic plants was performed by PCR, RT-PCR, SDS-PAGE, western blotting and zymography. Results of PCR showed that chimeric tPA was constructed using SOEing PCR. Analysis of transgenic plants using RT-PCR, SDS-PAGE and western blotting demonstrated that chimeric tPA is expressed. Moreover, zymography test showed that compared to normal tPA, produced chimeric tPA has more activity and stability. In conclusion increasing activity and stability of recombinant drugs using plant genes is promising.

Keywords

• chimeric
• cuscutain
• tissue plasminogen activator
• tobacco
• western blot
• zymography