HNOK Proteinlerinin Escherichia Coli’de Üretiminin Optimizasyonu

Yıl 2017, Cilt: 38 Ek Sayı 4, 86 - 97, 08.12.2017

Nur Başak Sürmeli

https://doi.org/10.17776/csj.363308

Öz

Hemoproteinler canlılarda steroid biyosentezinden solunuma,
sinyalizasyondan ilaç metabolizmasına kadar pek çok farklı biyolojik süreçlerde
önemli görevler üstlenirler. Endüstride, hemoproteinler kolesterol düşürücü
pravastatin, rahim ve rahim ağzı kanserlerinin hormonal tedavisinde kullanılan
progesteron, alerji ve yangı’ya karşı kullanılan kortizon gibi ilaçların
üretiminde kullanılmaktadır. Bunların yanı sıra hemoproteinlerin ilaç
geliştirilmesi, biyolojik iyileştirme gibi alanlarda kullanılması da planlanmaktadır.
Moleküler biyoloji ve protein tasarımı tekniklerinin gelişmesi ile bu
proteinlerin endüstriyel uygulama alanları da genişleyecektir. Hemoproteinlerin
bu alanlarda yaygın kullanımı karşısındaki en önemli engellerden biri Hem
kofaktörüne bağlı bir şekilde yüksek miktarda hemoprotein üretilememesidir. Bu
çalışmada Hem kofaktörüne bağlı bir şekilde üretilen hemoprotein miktarını en
yüksek seviyeye getirebilecek koşullar araştırılmıştır. Bu çalışma kapsamında
bakteride hemoprotein üretimini etkileyen en önemli üç etken olan indükleyici
izopropil β-D-l-tiyogalaktopiranosid (IPTG), ve Hem öncül molekülü
δ-aminolevülinik asit (ALA), ve ekspresyon sıcaklığı incelenmiştir. Bu
etkenlerin termofilik hemoprotein TtHNOK
üretimine etkisi araştırılmıştır. Özellikle, ALA pahalı bir molekül olduğu için
hemoproteinlerin üretiminde kullanılan ALA miktarının optimizasyonu önemlidir.
Bu çalışma sonucunda Escherichia coli
bakterisinde Hem kofaktörüne bağlı TtHNOK proteinin üretimi için en uygun
koşulların düşük sıcaklık, 0,5 mM IPTG ve 1 mM ALA olduğu gösterilmiştir. Bu
çalışmada çıkan sonuçlar Hem kofaktörüne bağlı hemoprotein üretiminde ALA
konsantrasyonun ve ekspresyon sıcaklığının önemli olduğunu göstermiştir. 

Anahtar Kelimeler

Hemoproteinler, protein ekspresyonu, hem biyosentezi, δ-aminolevülinik asit

Optimization of Hnox Protein Production in Escherichia Coli

Öz

Hemeproteins
carry a variety of different functions in organisms ranging from steroid
biosynthesis to respiration, signaling to drug metabolism. In industry,
hemeproteins are used for production of drugs such as: pravastatin for lowering
cholesterol, progesterone for hormonal treatment of cancers of uterus and
cervix, and cortisone used against allergy and inflammation. Hemeproteins can
also be used in drug development and biological remediation. The industrial
applications of hemeproteins will expand with development of molecular biology
and protein design techniques. One of the obstacles to the widespread use of
hemeproteins is difficulties in production of high levels of heme bound
protein. This study aims to maximize the amount of heme cofactor bound hemeprotein
produced. Here, three important factors affecting hemeprotein production in
bacteria are examined: induction by isopropyl β-D-1-thiogalactopyranoside
(IPTG), δ-aminolevulinic acid (ALA), precursor for heme biosynthesis, and
expression temperature. Effects of these factors on production of thermophilic
hemeprotein TtHNOX are investigated.
Since ALA is an expensive molecule, optimization of the amount of ALA used is
important. The most suitable conditions to produce TtHNOX is at low temperature, 0.5 mM IPTG and 1 mM ALA. This study
concludes that ALA concentration and expression temperature are important in
production of heme bound hemeproteins.

Anahtar Kelimeler

Hemeproteins, protein expression, heme biosynthesis, δ-aminolevulinic acid

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