Production of reverse transcriptase from Moloney murine Leukemia virus in Escherichia coli expression system

Yudhi Nugraha, Fina Amreta Laksmi, Isa Nuryana, Helbert & Firyal Nida Khasna

Abstract

Reverse transcriptase (RT) is one of the most important enzymes used in molecular biology applications, enabling the conversion of RNA into complementary DNA (cDNA) that is used in reverse transcription-polymerase chain reaction (RT-PCR). The high demand for RT enzymes in biotechnological applications makes the production optimization of RT crucial for meeting the growing demand in industrial settings. Conventionally, the expression of recombinant RT is a T7-induced promoter using IPTG in Escherichia coli expression systems, which is not cost-efficient. Here, we successfully made an alternative procedure for RT expression from Moloney murine leukemia virus (M-MLV) using the autoinduction method in a chemically defined medium. The optimization of carbon source composition (glucose, lactose, and glycerol) was analyzed using Response Surface Methodology (RSM). M-MLV RT was purified for further investigation of its activity. A total of 32.8 mg/L purified M-MLV RT was successfully obtained when glucose, glycerol, and lactose were present at concentrations of 0.06%, 0.9%, and 0.5%, respectively, making a 3.9-fold improvement in protein yield. In addition, the protein was produced in its active form by displaying 7462.50 U/mg of specific activity. This study provides the first step of small-scale procedures of M-MLV RT production that make it a cost-effective and industrially applicable strategy.