Cloning and Expression of Serotype-2 Simian Betaretrovirus Reverse Transcriptase Gene Isolated from Indonesian Cynomolgus Monkey in Escherichia coli

Authors

  • UUS SAEPULOH PRC IPB
  • DIAH ISKANDRIATI
  • FUNGKEY HOETAMA
  • SELA SEPTIMA MARIYA
  • DEDY DURYADI SOLIHIN
  • JOKO PAMUNGKAS
  • DONDIN SAJUTHI

DOI:

https://doi.org/10.5454/mi.7.2.3

Keywords:

Escherichia coli expression system, recombinant enzyme, reverse transcriptase, SRV-2

Abstract

In this study, we isolated the simian betaretrovirus serotype-2 (SRV-2) reverse transcriptase (RT) gene from infected Indonesian cynomolgus monkey (Macaca fascicularis). The gene was then cloned in Escherichia coli expression system. The SRV-2 RT gene is located between nucleotides 3284-4925 in the polyprotein (Pol) region encodes 547 amino acids. Analysis of expression using SDS-PAGE and western blot techniques showed a specific band of 64.9 kDa, indicating SRV-2 RT recombinant enzyme. Purification of SRV-2 RT recombinant enzyme produced 312 μg mL-1 protein with 7.1 U μL-1 enzyme activities. Application of this recombinant enzyme in reverse transcription-PCR (RT-PCR) of β-globin and β-actin genes produced DNA fragments of 206 and 350 bp, indicating amplification of β-globin and β-actin genes, respectively. Therefore, the expressed SRV-2 RT enzyme was proven to be functional, although the activity was low.

Published

2013-06-30

How to Cite

SAEPULOH, U., ISKANDRIATI, D., HOETAMA, F., MARIYA, S. S., SOLIHIN, D. D., PAMUNGKAS, J., & SAJUTHI, D. (2013). Cloning and Expression of Serotype-2 Simian Betaretrovirus Reverse Transcriptase Gene Isolated from Indonesian Cynomolgus Monkey in Escherichia coli. Microbiology Indonesia, 7(2), 3. https://doi.org/10.5454/mi.7.2.3

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