Synthesis of the human insulin gene: protein expression, scaling up and bioactivity.
Optimized Synthetic human insulin gene was preferred to easy of cloning, plasmid stability, and protein expression away from the native sequence and its rare codons. Two steps to obtain the insulin, so we assembled the gene of 293 bp using a battery of overlapped synthetic oligos, then cloned into pET101directional TOPO expression vector downstream to the T7 promoter. The proinsulin products were produced as inclusion bodies in E. coli at a level of 10%. The batch cultivation of the strain yielded 6 g/L, while the high cell density of fed-batch cultivation yielded 46 g/L. The proinsulin purification yielded 110 mg/gram cell weight, and 1.3 mg/gram of a bioactive insulin. The native insulin was generated by enzymatic conversion of chemically processed proinsulin. The produced insulin was matched with that of a commercial aqueous version at a level of enzyme immunoassys, SDS-PAGE, RP-HPLC, and bioactivity. The present results showed that the produced insulin has a comparable biochemical and potency similar to that of commercial one.
Redwan
El-Rashdy M
el-RM
Antibody Laboratory, Protein Research, Genetic Engineering and biotechnology Research Institute, Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt. redwan1961@yahoo.com
Matar
Saleh M
SM
El-Aziz
Gamal Abd
GA
Serour
Ehab A
EA
D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007118:Immunoassay; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
D003470:Culture Media; D007328:Insulin