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

被引:3
作者
Nugraha, Yudhi [1 ]
Laksmi, Fina Amreta [2 ]
Nuryana, Isa [2 ]
Helbert [2 ]
Khasna, Firyal Nida [2 ]
机构
[1] Natl Res & Innovat Agcy, Res Ctr Mol Biol Eijkman, Jalan Raya Bogor Km 4, Bogor 16911, West Java, Indonesia
[2] Natl Res & Innovat Agcy, Res Ctr Appl Microbiol, Bogor, West Java, Indonesia
关键词
Autoinduction; chemically defined medium; Escherichia coli; Moloney murine Leukemia virus; reverse transcriptase; DEPENDENT DNA-POLYMERASE; HIGH-LEVEL PRODUCTION; AUTOINDUCTION; PROTEIN; RECOMBINANT; CULTURE; VIRIONS; INDUCER; DOMAIN; RT;
D O I
10.1080/10826068.2024.2317311
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 of RT enzymes in biotechnological applications making the production optimization of RT is crucial for meeting the growing demand in industrial settings. Conventionally, the expression of recombinant RT is 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 autoinduction method in 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 on its activity. A total of 32.8 mg/L purified M-MLV RT was successfully obtained when glucose, glycerol, and lactose were present at concentration 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.
引用
收藏
页码:1079 / 1087
页数:9
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