İnsan İnsülin Hormonu Öncülerinin Pichia pastoris AOX1 Promotoru Altında Klonlanması, Ekspresyonu ve Biyoreaktörde Üretimi

Abstract

İnsülin pankreasın beta hücrelerinden üretilen ve vücutta glukoz dengesini sağlayan önemli bir peptit hormondur. Pankreasın yeterli miktarda insülin üretememesi ya da hücrelerin üretilen insüline cevap verememesi sonucu kan glukoz düzeyinin yükselmesiyle diyabet adı verilen metabolik bir hastalık meydana gelir. Günümüzde bu hastalığın tedavisinde insülin hormonu kullanılmaktadır. İnsülin hormonu genetik mühendisliği teknikleri kullanılarak rekombinant olarak üretilen ilk proteindir. İlk olarak Escherichia coli ve Saccharomyces cerevisiae’da üretilmeye başlanmış, ancak son zamanlarda rekombinant insülin üretiminde Pichia pastoris (Komagataella phaffi) ’in  kullanımı yaygınlaşmıştır. Bu çalışmada, insan insülin hormonu öncülerinin (IP) P. pastoris’in metanol ile indüklenebilir AOX1 promotoru altında üretimini sağlamak amacıyla; bu proteini kodlayan DNA fragmenti transformasyon ve ligasyon gibi moleküler biyoloji teknikleri kullanılarak plazmide aktarılarak istenen proteini kodlayan bir ekspresyon vektörü elde edilmiştir. Ekspresyon vektörünün lityum asetat yöntemiyle yetenekli hale getirilen P. pastoris X33 suşuna elektroporasyonla transferi sağlanmıştır. 5L ölçekli biyoreaktörde yapılan protein ekspresyonu çalışması sonrasında alınan örnekler SDS-PAGE ve ELISA yöntemleriyle analiz edilmiş ve 7.5 mg/L IP proteini üretildiği tespit edilmiştir.

Keywords

• İnsülin,Pichia pastoris,AOX1 promotoru,Rekombinant protein

Cloning, Expression and Production in a Bioreactor of Human Insulin Precursor in Pichia pastoris under AOX1 Promoter

Abstract

Insulin is a peptide hormone, produced by beta cells of the pancreas, and regulates the glucose homeostasis in the body. Diabetes mellitus is a metabolic disease in which a person has high blood sugar, either because the pancreas does not produce enough insulin, or because cells do not respond to produced insulin. At the present time, insulin is used to treatment of this disease. Insulin hormone is first protein produced recombinantly by using genetic engineering techniques. Firstly, it was produced in E. coli and Saccharomyces cerevisiae, but recently the number of studies that used Pichia pastoris (Komagataella phaffi) in the production of recombinant insulin increase. In this study, in order to produce human insulin hormone precursor (IP) under the  inducible AOX1 promoter in P. pastoris, an expression vector encoding desired protein was obtained by transferring DNA fragment coding this protein to the plasmid using molecular biology techniques such as transformation and ligation. Expression vector was transferred via electroporation to Pichia pastoris  X33 strain which rendered by lithium acetate method. After protein expression study in 5L bioreactor, obtained samples were analyzed with SDS-PAGE and ELISA methods and determined 7.5 mg/L protein produced.

Keywords

• Insulin,Pichia pastoris,AOX1 promoter,Recombinant protein

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