Applied Biochemistry and Biotechnology

, Volume 169, Issue 1, pp 77–87

Enzymatic Characterization of Human Immunodeficiency Virus Type 1 Reverse Transcriptase for Use in cDNA Synthesis

  • Atsushi Konishi
  • Mayu Shinomura
  • Kiyoshi Yasukawa
Article

DOI: 10.1007/s12010-012-9953-8

Cite this article as:
Konishi, A., Shinomura, M. & Yasukawa, K. Appl Biochem Biotechnol (2013) 169: 77. doi:10.1007/s12010-012-9953-8

Abstract

The aim of this study is to explore the advantages of using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in cDNA synthesis. Recombinant HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT were produced in an Escherichia coli expression system. In the incorporation of dTTP into poly(rA)-p(dT)15 (T/P), the Km values for dTTP of HIV-1 M RT and HIV-1 O RT were 8 and 12 % of that of Moloney murine leukemia virus (MMLV) RT, respectively, and the Km values for T/P were 25 and 23 % of that of MMLV RT, respectively. Compared with MMLV RT, HIV-1 M RT and HIV-1 O RT were less susceptible to formamide, which is frequently used for cDNA synthesis with a G + C-rich RNA to improve specificity. The high substrate affinity and low susceptibility to formamide of HIV-1 RT might be advantageous for its use in cDNA synthesis.

Keywords

cDNAFormamideHIV-1MMLVReverse transcriptase
DMSO

Dimethyl sulfoxide

HIV-1

Human immunodeficiency virus type 1

HIV-1 M

HIV-1 group M

HIV-1 O

HIV-1 group O

MMLV

Moloney murine leukemia virus

PMSF

Phenylmethylsulfonyl fluoride

RT

Reverse transcriptase

T/P

Template–primer

Supplementary material

12010_2012_9953_MOESM1_ESM.pdf (44 kb)
Fig. S1(PDF 43 kb)
12010_2012_9953_MOESM2_ESM.doc (58 kb)
ESM Tables(DOC 58 kb)

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Atsushi Konishi
    • 1
  • Mayu Shinomura
    • 1
  • Kiyoshi Yasukawa
    • 1
  1. 1.Division of Food Science and BiotechnologyGraduate School of Agriculture, Kyoto UniversityKyotoJapan