PURIFICATION OF SOLUBLE OVER-EXPRESSED HUMAN GROWTH HORMONE(TRX-HIS6-HGH) IN ESCHERICHIA COLI WITH NI-NTA CHROMATOGRAPHY SHOWED CLASSI NATIVE PROTEIN CONTAMINANTS

Background and Aim:Human growth hormone(hGH) is a 22kDa polypeptide consists of 191 amino acids and two disulfide bonds. This hormone has therapeutic applications. E.coli is the preferred host since hGH has no need to post-translational modification. Often Trx, NusA, FH8 tags are used to express soluble protein in E.coli. By using Ni-NTA chromatography very little amount of the target protein in the protein mixture can be purified with high purity in just one step. In this study Trx-His6-hGH was purified with Ni-NTA chromatography.Methods:The gene cassette was designed. It consists of, from 5 to 3, Trx, His6, enterokinase and hGH respectively. The cassette is first cloned in the pET-32a(+) plasmid and then transferred to E.coli DH5 and afterward to E.coli Rosettagami and expression was performed in Terrific Broth(TB) in 25C. Then cleared cell lysate was applied to Ni-NTA column. The purification was done on imidazole concentrations of 5mM, 20mM and 50mM and the results were analyzed by SDS-PAGE.Results:SDS-PAGE results showed no purity in purification with 5mM of imidazole in equilibration buffer. The purity was approved with 20mM but there were some proteins that co-purified with the Trx-His6-hGH at the elution step. The purity was significantly approved with 50mM and protein contaminants were decreased significantly. The average purity of purified Trx-His6-rhGH was 86% with the yield of 37.5%.Conclusion:According to the results, purification of over-expressed soluble hGH in E.coli can be achieved appropriately by using His6 tag and Ni-NTA chromatography. The protein contaminants co-purified in this study belongs to worst class(Class I).

کلیدواژه ها:

Recombinant Human Growth Hormone ، rhGH ، Trx-His6-hGH ، Ni-NTA Chromatography ، ClassI native protein contaminants in His6-hGH Purification ، Purification of Trx-His6-hGH using Ni-NTA Chromatography