Optimization of the apolipoprotein B mRNA editing enzyme catalytic polypeptidelike-3G (APOBEC3G) gene to enhance its expression in Escherichia coli

  • Rizkyana Avissa Master Program of Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
  • Silvia Tri Widyaningtyas Virology and Cancer Pathobiology Research Center, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia https://orcid.org/0000-0001-6439-5943
  • Budiman Bela Virology and Cancer Pathobiology Research Center, Faculty of Medicine, Universitas Indonesia, Cipto Mangunkusumo Hospital, Jakarta, Indonesia; Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia https://orcid.org/0000-0002-9010-2427
Keywords: APOBEC3G, codon usage, prokaryote gene expression
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Abstract

BACKGROUND Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like-3G (APOBEC3G) can abolish HIV infection by inducing lethal mutations in the HIV genome. The HIV protein virion infectivity factor (Vif) can interact with APOBEC3G protein and cause its degradation. Development of a method that can screen substances inhibiting the APOBEC3G-Vif interaction is necessary for identification of substances that potentially used in anti-HIV drug development. In order to increase expression of recombinant APOBEC3G protein that will be used in APOBEC3G-Vif interaction assay, we developed an optimized APOBEC3G gene for expression in Escherichia coli

METHODS The gene coding APOBEC3G was codon-optimized in accordance with prokaryotic codon using DNA 2.0 software to avoid bias codons that could inhibit its expression. The APOBEC3G gene was synthesized and sub-cloned into pQE80L plasmid vector. pQE80L containing APOBEC3G was screened by polymerase chain reaction, enzyme restriction, and sequencing to verify its DNA sequence. The recombinant APOBEC3G was expressed in E. coli under isopropyl-β-D-thiogalactoside (IPTG) induction and purified by using nickel-nitrilotriacetic acid (Ni-NTA) resin. 

RESULTS The synthetic gene coding APOBEC3G was successfully cloned into the pQE80L vector and could be expressed abundantly in E. coli BL21 in the presence of IPTG. 

CONCLUSIONS Recombinant APOBEC3G is robustly expressed in E. coli BL21, and the APOBEC3G protein could be purified by using Ni-NTA. The molecular weight of the recombinant APOBEC3G produced is smaller than the expected value. However, the protein is predicted to be able to interact with Vif because this interaction is determined by a specific domain located on the N-terminal of APOBEC3G. 

References

  1. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK, Watt IN, et al. DNA deamination mediates innate immunity to retroviral infection. Cell. 2003;113(6):803-9. https://doi.org/10.1016/S0092-8674(03)00423-9

  2. da Costa KS, Leal E, dos Santos AM, Lima E Lima AH, Alves CN, Lameira J. Structural analysis of viral infectivity factor of HIV type 1 and its interaction with APOBEC3G, EloC and EloB. PLoS One. 2014;9(2):e89116. https://doi.org/10.1371/journal.pone.0089116

  3. Nathans R, Cao H, Sharova N, Ali A, Sharkey M, Stranska R, et al. Small-molecule inhibition of HIV-1 Vif. Nat Biotechnol. 2008;26(10):1187-92. https://doi.org/10.1038/nbt.1496

  4. Polevoda B, McDougall WM, Bennett RP, Salter JD, Smith HC. Structural and functional assessment of APOBEC3G macromolecular complexes. Methods. 2016;107(1):10-22. https://doi.org/10.1016/j.ymeth.2016.03.006

  5. Iwatani Y, Takeuchi H, Strebel K, Levin JG. Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect. J Virol. 2006;80(12):5992-6002. https://doi.org/10.1128/JVI.02680-05

  6. Li L, Yang YS, Li ZL, Zeng Y. Prokaryotic expression and purification of HIV-1 Vif and hAPOBEC3G, preparation of polyclonal antibodies. Virol Sin. 2008;23(3):173-82. https://doi.org/10.1007/s12250-008-2909-z

  7. Oliver JL, Marín A. A relationship between GC content and coding-sequence length. J Mol Evol. 1996;43(3):216-23. https://doi.org/10.1007/BF02338829

  8. Sharpl PM, Li WH. The codon adaptation index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987;15(3):1281-95. https://doi.org/10.1093/nar/15.3.1281

  9. Muto A, Osawa S. The guanine and cytosine content of genomic DNA and bacterial evolution. Proc Natl Acad Sci U S A. 1987;84(1):166-9. https://doi.org/10.1073/pnas.84.1.166

  10. Qiagen. QIAEX® II Handbook for DNA extraction from agarose and polyacrilamide gels and for desalting and concentrating DNA from solutions. 2015.

  11. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870-4. https://doi.org/10.1093/molbev/msw054

  12. Jónsson SR, Andrésdóttir V. Host restriction of lentiviruses and viral countermeasures: APOBEC3 and Vif. Viruses. 2013;5(8):1934-47. https://doi.org/10.3390/v5081934

  13. Zennou V, Bieniasz PD. Comparative analysis of the antiretroviral activity of APOBEC3G and APOBEC3F from primates. Virology. 2006;349(1):31-40. https://doi.org/10.1016/j.virol.2005.12.035

  14. Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM. Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci U S A. 2009;106(6):1760-5. https://doi.org/10.1073/pnas.0813167106

  15. Iakoucheva LM, Kimzey AL, Masselon CD, Smith RD, Dunker AK, Ackerman EJ. Aberrant mobility phenomena of the DNA repair protein XPA. Protein Sci. 2001;10(7):1353-62. https://doi.org/10.1110/ps.40101

  16. Guan Y, Zhu Q, Huang D, Zhao S, Lo LJ, Peng J. An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide. Sci Rep. 2015;5:13370. https://doi.org/10.1038/srep13370

Published
2020-06-30
How to Cite
1.
Avissa R, Widyaningtyas ST, Bela B. Optimization of the <em> apolipoprotein B mRNA editing enzyme catalytic polypeptidelike-3G </em> (<em>APOBEC3G</em>) gene to enhance its expression in <em> Escherichia coli </em&gt;. Med J Indones [Internet]. 2020Jun.30 [cited 2020Aug.7];29(2):120-8. Available from: https://mji.ui.ac.id/journal/index.php/mji/article/view/2853
Section
Basic Medical Research