Ekspresi Enzim Rekombinan Reverse Transcriptase (RTRNase H) Simian Betaretrovirus Serotipe-2 Asal Macaca fascicularis Indonesia dalam Sistem Ekspresi Eschericia coli

Uus Saepuloh, Diah Iskandriati, Joko Pamungkas, Dondin Sajuthi

Abstract


Reverse transcriptase (RT) enzyme is a key tool in molecular biology for the synthesis of complementary DNA (cDNA) from messenger RNA (mRNA). Combining RT activity with PCR amplification has been a gold standard as the first step in cloning the coding region of any gene of interest. Evidently, RTs have been critical in advancing molecular biology, genetics and medicine to their current stage. In this study, we were developing the RTDRNase H recombinant enzyme isolated from serotype-2 simian betaretrovirus-2 (SRV-2) infected Indonesian Macaca fascicularis using Escherichia coli expression system. The study was conducted using RT SRV-2 gene expression using E. coli expression system, proteins purification, and application to RT PCR technique. The SDS PAGE expression analysis showed a specific band size of 32.7 kDa assumed as RT protein enzyme. Application of RT SRV-2 enzyme generated to the RT PCR technique of β-globin CDV and SRV-2 env gene target showed that the RT SRV-2 enzyme was capable to reverse transcribed mRNA into cDNA as indicated by the presence of specific DNA band compared with commercial RT enzymes. This RT SRV-2 enzyme showed its activity similar to that of commercial one, although the activity was lower.


Keywords


escherichia coli expression system; recombinant enzyme; reverse transcriptase; SRV-2

Full Text:

PDF

References


Chen Y, Xu W, Sun Q. 2009. A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Eschericia coli. Biotechnol Lett. 31: 1051-1057.

Flint SJ, Enquist LW, Racaniello VR, Skalka AM. 2009. Principle of Virology: Molecular Biology, Pathogenesis, and Control. Washington DC (US): ASM Pr.

Herschhorn A, Hizi A. 2010. Retroviral reverse transcriptase. Cell Mol Life Sci. 67(16): 2717-2747.

Hizi A, Herschhorn A. 2007. Retroviral reverse transcriptases (other than those of HIV-1 and murine leukemia virus): A comparison of their molecular and biochemical properties. Virus Res. 134(1-2): 203-220.

Iskandriati D, Saepuloh U, Mariya S, Grant RF, Solihin DD, Sajuthi D, Pamungkas J. 2010. Isolation and characterization of simian retrovirus type D from Macaca fascicularis and M. nemestrina in Indonesia. Microbiol Indones. 4(3): 132-136.

Kohlstaedt LA, Wang J, Friedman JM, Rice PA, Steitz TA. 1992. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 256(5065): 1783-1790.

Lerche NW. 2010. Simian retroviruses: Infection and disease-implications for immunotoxicology research in primates. J Immunotoxicol. 7(2): 93-101.

Marracci GH, Kelley RD, Pilcher KY, Crabtree L, Shiigi SM, Avery N, Leo G, Webb MC, Hallick LM, Axthelm MK. 1995. Simian AIDS type D serogroup 2 retrovirus: isolation of an infectious molecular clone and sequence analyses of its envelope glycoprotein gene and 3' long terminal repeat. J Virol. 69(4): 2621-2628.

Marracci GH, Avery NA, Shiigi SM, Couch G, Palmer H, Pilcher KY, Nichols H, Hallick LM, Axthelm MK, Machida CA. 1999. Molecular cloning and cell-specific growth characterization of polymorphic variants of type D serogroup 2 simian retroviruses. Virol. 261(1): 43-58.

Nandi JS, Van DS, Chhangani AK, Mohnot SM. 2006. New simian ß retroviruses from rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) from Rajasthan, India. Virus Genes. 33(1): 107-116.

Philipp-Staheli J, Marquardt T, Thouless ME, Bruce AG, Grant RF, Tsai CC, Rose TM. 2006. Genetic variability of the envelope gene of Type D simian retrovirus-2 (SRV-2) subtypes associated with SAIDS-related retroperitoneal fibromatosis in different macaque species. Virol J. 3: 11-25.

Polumuri SK, Ruknudin A, Schulze DH. 2002. RNase H and its effects on PCR. Biotechniques. 32(6): 1224-1225.




View JIPI Stats

 

   Creative Commons License

This journal is published under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License.

Editorial Office: Institute for Research and Community Services (LPPM), Andi Hakim Nasoetion Building, 5th Floor, Jl. Raya Darmaga, IPB Darmaga Campus, Bogor, West Java, Indonesia 16680, Telp/Fax: +62251-8622323, email: jipi-lppm@apps.ipb.ac.id; jipi-lppm@ipb.ac.id