Invitro cytotoxicity study on recombinant human insulin produced in pichia pastoris

Background: Recombinant DNA technology has made a revolutionary impact in the area of human healthcare by enabling mass production of safe, pure and effective rDNA expression products, currently several categories of rDNA products like hormones of therapeutic interest, haemopoietic growth factors, blood coagulation products, thrombolytic agents, anticoagulants, interferons, interleukins and therapeutic enzymes are being produced using recombinant DNA technology for human use. Materials and Methods :Production of recombinant human insulin was perfumed by standard methods total RNA was isolated from pancreatic tissue, then RNA was converted to cDNA using RT-P RCR reaction and preproinsulin gene was amplified using specific primers and inserted into the EcoRI and NotI sites of pPIC9K to produce the expression plasmid named pPIC9K-hpi expression vector . Which then transferred to Pichia pastoris by electroporation method? MTT assay and hemolysis test were carried out to evaluated the invitro cytotoxicity of recombinant human insulin that expressed into Pichia pastoris . Results: the results demonstrate that the different tested concentrations of recombinant human insulin showed no inhibitory effect on the metabolism activity and cell viability of the human blood lymphocytes after a 24 h exposure, furthermore the result shown that the human insulin had no hemolytic activity against the human red blood cells within a different concentration used. Conclusions: overall results amply demonstrate that the human insulin produced in this work is safe and had no cytotoxicity activity as the standard formulated insulin and recommended as hypoglycemic medical drug.