PURIFICATION OF SOLUBLE OVER-EXPRESSED HUMAN GROWTH HORMONE(TRX-HIS6-HGH) IN ESCHERICHIA COLI WITH NI-NTA CHROMATOGRAPHY SHOWED CLASSI NATIVE PROTEIN CONTAMINANTS
- سال انتشار: 1397
- محل انتشار: نوزدهمین کنگره بین المللی میکروب شناسی ایران
- کد COI اختصاصی: MEDISM19_364
- زبان مقاله: انگلیسی
- تعداد مشاهده: 424
نویسندگان
Seyed Mohammad Mehdi Hasanpour Matikolaee
Department of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
Department of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
Department of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
Department of Science and Biotechnology, Malek Ashtar University of Technology, Tehran, Iran
چکیده
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 Chromatographyاطلاعات بیشتر در مورد COI
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