High-Level Production of MMLV Reverse Transcriptase Enzyme in Escherichia Coli

Abstract

Moloney murin lösemi virüs (MMLV) ters transkriptazı (RT), cDNA sentezi ve RNA amplifikasyonu için en yaygın kullanılan enzimdir. Bu çalışmada moleküler çalışmalardaki önemi nedeniyle MMLV RT enziminin rekombinant olarak üretilmesi amaçlanmıştır. Bu bağlamda, MMLV RT enzimini kodlayan DNA fragmanı pTOLT plazmidine klonlanmış ve E. coli BL21 (DE3) pLysE hücrelerinde eksprese edilmiştir. Proteinin yüksek düzeyde ekspresyonu, protein moleküllerinin inklüzyon cisimciklerinde toplanmasına neden olduğundan MMLV RT proteininin çözünür formda elde edilebilmesi için MMLV RT ve şaperon plazmidlerin (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) birlikte ekspresyonu gerçekleştirilmiştir. Beklentilerimizin aksine, protein çözünür formda elde edilemediği için, yeniden katlama prosesi kullanılarak inklüzyon cisimciklerinden geri kazanılmıştır. Son olarak protein, afinite kromatografisiyle saflaştırılmış ve proteinin aktivitesi RT-PCR tekniği kullanılarak kontrol edilmiştir.

Keywords

• İnklüzyon cisimcikleri
• Moloney murin lösemi virüsü
• Ters transkriptaz
• cDNA sentezi

High-Level Production of MMLV Reverse Transcriptase Enzyme in Escherichia Coli

Abstract

Reverse transcriptase (RT) of Moloney murine leukemia virus (MMLV) is the most widely used enzyme for cDNA synthesis and RNA amplification. In this study, we aimed to produce MMLV RT enzyme recombinantly due to its importance in molecular studies. In this context, the DNA fragment encoding the MMLV RT enzyme was cloned into pTOLT plasmid and expressed in E. coli BL21 (DE3) pLysE cells. Since the high-level expression of the protein caused the protein molecules to aggregate in the inclusion bodies, co-expression of MMLV RT and chaperone plasmids (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) was performed to obtain the MMLV RT protein in soluble form. Contrary to our expectations, because it could not be obtained in soluble form, the protein was recovered from the inclusion bodies using refolding process. Finally, the protein was purified by affinity chromatography and the activity of the protein was checked using RT-PCR technique.

Keywords

• Inclusion bodies
• Moloney murine leukemia virus
• Reverse transcriptase
• cDNA synthesis

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