Soluble Expression of Human Granulocyte Colony Stimulating Factor (hG-CSF) in Escherichia coli Expression System

Yıl 2025, Cilt: 15 Sayı: 3, 960 - 970, 15.09.2025

Mustafa Songur , Özlem Kaplan , Rizvan İmamoğlu , İsa Gökçe

https://doi.org/10.31466/kfbd.1465411

Öz

Human granulocyte colony stimulating factor (hG-CSF) is a hematological growth factor that plays a crucial role in neutrophil production and differentiation. Some foreign biomolecules, especially of human origin, such as hG-CSF, sometimes aggregate because of different factors during expression and create inclusion bodies in Escherichia coli (E. coli). Refolding process is commonly used to recover these very valuable molecules, but still significant amounts of protein remain unusable. Refolding procedures are frequently costly, time-consuming, and not fully efficient. Therefore, the use of molecular chaperones to improve soluble expression of proteins was evaluated in the study. In this context, hG-CSF was co-expressed with five chaperone plasmid systems (pGro7, pG-KJE8, pG-Tf2, pKJE7, pTf16) to ensure the expression of hG-CSF in soluble form. Among these, the pKJE7 plasmid was found to be the most effective in obtaining hG-CSF in soluble form, yielding 92% purity after Ni-NTA affinity chromatography purification. The total yield of hG-CSF obtained was 1.6 mg per 1 L bacterial culture. The biological activity of the soluble hG-CSF was evaluated in human umbilical vein endothelial cells (HUVECs). A 24-hour interaction of hG-CSF with HUVECs resulted in a significant increase in cell viability at all applied doses, demonstrating its bioactivity. As a result, hG-CSF, which previously aggregated as an inclusion body in the E. coli expression system, was correctly folded by co-expression with chaperone proteins were obtained as more efficient and purer.

Anahtar Kelimeler

Granulocyte colony stimulating factor , Recombinant proteins , Molecular chaperones , E. coli

Proje Numarası

2020/67

Escherichia coli İfade Sisteminde İnsan Granülosit Koloni Uyarıcı Faktörün (hG-CSF) Çözünür İfadesi

Öz

İnsan granülosit koloni uyarıcı faktör (hG-CSF), nötrofil üretimi ve farklılaşmasında çok önemli bir rol oynayan hematolojik bir büyüme faktörüdür. hG-CSF gibi özellikle insan kökenli bazı yabancı biyomoleküller bazen ekspresyon sırasında farklı faktörler nedeniyle bir araya gelir ve Escherichia coli'de (E. coli) inklüzyon cisimcikleri oluşturur. Bu çok değerli molekülleri geri kazanmak için yeniden katlama işlemi yaygın olarak kullanılır, ancak yine de önemli miktarda protein kullanılamaz durumda kalır. Yeniden katlama prosedürleri sıklıkla maliyetlidir, zaman alıcıdır ve tam olarak verimli değildir. Bu nedenle çalışmada proteinlerin çözünür ifadesini geliştirmek için moleküler şaperonların kullanımı değerlendirildi. Bu bağlamda, hG-CSF'nin çözünür formda ekspresyonunu sağlamak için hG-CSF, beş şaperon plazmit sistemi (pGro7, pG-KJE8, pG-Tf2, pKJE7, pTf16) ile birlikte eksprese edildi. Yapılan deneyler sonucunda, pKJE7 plazmitinin hG-CSF’nin çözünür formda elde edilmesinde en etkili sistem olduğu belirlenmiş ve Ni-NTA afinite kromatografisi ile %92 saflıkta saflaştırılmıştır. Elde edilen hG-CSF'nin toplam verimi, 1 L'lik bakteri kültüründen 1,6 mg olarak hesaplanmıştır. Çözünür formdaki hG-CSF’nin biyolojik aktivitesi, insan göbek kordonu endotel hücreleri (HUVECs) üzerinde değerlendirilmiş ve 24 saatlik etkileşim sonucunda, tüm uygulanan dozlarda hücre canlılığında anlamlı bir artış sağladığı gözlemlenmiştir. Sonuç olarak daha önce E. coli ekspresyon sisteminde inklüzyon cisimciği olarak agregasyona uğrayan hG-CSF'nin şaperon proteinleri ile ekspresyonu yapılarak doğru bir şekilde katlanmasıyla daha verimli ve daha saf bir şekilde elde edildi.

Anahtar Kelimeler

Granülosit koloni uyarıcı faktör , Rekombinant proteinler , Moleküler şaperonlar , E. coli

Destekleyen Kurum

Tokat Gaziosmanpaşa University

Proje Numarası

2020/67

Teşekkür

The study was funded by Tokat Gaziosmanpaşa University, Foundation of Scientific Research Projects (Project number: 2020/67).

Kaynakça

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