Method Optimization of Reverse Phase High Performance Liquid Chromatography (RP-HPLC) for Characterization of Digested Products of Recombinant Pro-Insulin

Recombinant insulin has been produced by expressing multiple joined pro-insulin genes in E.coli. Refolded pro-insulin has been converted to insulin upon brief reactions with trypsin and carboxypeptidase-B. Finally, the bioactive insulin is separated by RP-HPLC (Mukhopadhyay, 1997, Marks, 1989). In this study, the effect of several parameters on RP-HPLC of recombinant pro-insulin was investigated. These parameters were TFA concentration (0.05-0.2 %), temperature (room temperature up to 500C), gradient (0-100% acetonitrile in different times) and flow rate (0.6-1 ml/min). We suggested the optimum gradient for separation and characterization of digested products of pro-insulin especially insulin and arg-insulin using 0-100% acetonitrile with 0.1% TFA in 80 minute at flow rate of 0.8 ml/min in 45C.

کلیدواژه ها:

HPLC ، Chromatography ، Insulin ، Arg-insulin