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

Year 2018, Volume: 22 Issue: 3, 1190 - 1196, 20.09.2018

Aysun Türkanoğlu Özçelik Ayşe Torun Semiramis Yılmaz Mehmet İnan

https://doi.org/10.19113/sdufenbed.472329

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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