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, Institute of chemical Technology, India
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.
More details :- https://airccse.com/bioej/papers/2115bioej01.pdf
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