Bioscience & Engineering: An International Journal (BIOEJ)

CLONING AND EXTRACELLULAR EXPRESSION OF RECOMBINANT TISSUE PLASMINOGEN ACTIVATOR (RT-PA) USING A METHYLOTROPHIC YEAST PICHIA PASTORIS 

Atul M. Vhanmarathi, Rekha Matlani and Arvind M.Lali 

DBT-ICT-Centre for Biosciences, Institute of chemical Technology, Mumbai  

ABSTRACT 

Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration. The transformants were screened for phenotypic characters.Mut+ phenotypic colony named Pichia tPA-3 showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will surely provide cost effective alternative to mammalian system forrt-PA production. 

KEYWORDS 

Tissue plasminogen activator, acute myocardial infarctions, Pichia pastoris, pICZαA, FactorXa protease, SDS PAGE, Size Exclusion Chromatography etc. 

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