Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Directed Evolution of a β-Glucosidase for Improved Functions as a Reporter in Protein Expression

  • Lim, Ho-Dong (Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University) ;
  • Han, So-Young (Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University) ;
  • Park, Gi-Hye (Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University) ;
  • Cheong, Dae-Eun (Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University) ;
  • Kim, Geun-Joong (Department of Biological Sciences and Research Center of Ecomimetics, College of Natural Sciences, Chonnam National University)
  • Received : 2022.03.28
  • Accepted : 2022.04.18
  • Published : 2022.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

Precisely reliable and quantitative reporters can provide phenotypes that are consistent with research goals in protein expression. Here, we developed an improved reporter mATglu III 5 by directed evolution using a versatile β-glucosidase ATglu derived from Agrobacterium tumefaciens. When expressed in hosts, a vector containing this mutant distinctly showed a colored or fluorescent phenotype, according to the supplemented substrate, without any inducer. Analysis of mATglu III 5 showed it to be fully functional in fusion state with oligomeric proteins, especially under non-induction conditions, thereby offering an alternative to conventional reporters.

Keywords

Acknowledgement

This work was supported by a grant (grant number: 20170305) from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries, Korea. This work was also supported by a grant (grant number: 2021R1A2C1006734) of the Basic Science Research program of the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology of Korea (MEST), Republic of Korea.

References

  1. Naylor LH. 1999. Reporter gene technology: the future looks bright. Biochem. Pharmacol. 58: 749-757. https://doi.org/10.1016/S0006-2952(99)00096-9
  2. Jiang T, Xing B, Rao J. 2008. Recent developments of biological reporter technology for detecting gene expression. Biotechnol. Genet. Eng. Rev. 25: 41-75. https://doi.org/10.5661/bger-25-41
  3. Tsien RY. 1998. The green fluorescent protein. Annu. Rev. Biochem. 67: 509-544. https://doi.org/10.1146/annurev.biochem.67.1.509
  4. Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. 2006. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24: 79-88. https://doi.org/10.1038/nbt1172
  5. Shaner NC, Campbell RE, Steinbach PA, Giepmans BNG, Palmer AE, Tsien RY. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22: 1567-1572. https://doi.org/10.1038/nbt1037
  6. Shuman HA, Silhavy TJ. 2003. The art and design of genetic screens: Escherichia coli. Nat. Rev. Genet. 4: 419-431. https://doi.org/10.1038/nrg1087
  7. Iost I, Dreyfus M. 1995. The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation. EMBO J. 14: 3252-3261. https://doi.org/10.1002/j.1460-2075.1995.tb07328.x
  8. Wu N, Rathnayaka T, Kuroda Y. 2015. Bacterial expression and reengineering of Gaussia princeps luciferase and its use as a reporter protein. Biochim. Biophys. Acta 1854: 1392-1399. https://doi.org/10.1016/j.bbapap.2015.05.008
  9. Chernov KG, Redchuk TA, Omelina ES, Verkhusha VV. 2017. Nearinfrared fluorescent proteins, biosensors, and optogenetic tools engineered from phytochromes. Chem. Rev. 117: 6423-6446. https://doi.org/10.1021/acs.chemrev.6b00700
  10. Kim JY, Lee, JY, Shin YS, Kim GJ. 2009. Mining and identification of a glucosidase family enzyme with high activity toward the plant extract indican. J. Mol. Catal. B 57: 284-291. https://doi.org/10.1016/j.molcatb.2008.10.001
  11. Kim JY, Lee JY, Shin YS, Kim GJ. 2010. Characterization of an indican-hydrolyzing enzyme from Sinorhizobium meliloti. Process Biochem. 45: 892-896. https://doi.org/10.1016/j.procbio.2010.02.017
  12. Cheong DE, Chang WS, Kim GJ. 2012. A cloning vector employing a versatile β-glucosidase as an indicator for recombinant clones. Anal. Biochem. 425: 166-168. https://doi.org/10.1016/j.ab.2012.03.004
  13. Han SS, Park WJ, Kim HS, Kim GJ. 2015. Antisense RNA-based high-throughput screen system for directed evolution of quorum quenching enzymes. ACS Chem. Biol. 10: 2598-2605. https://doi.org/10.1021/acschembio.5b00714
  14. Park WJ, You SH, Choi HA, Chu YJ, Kim GJ. 2015. Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in Escherichia coli. Acta Biochim. Biophys. Sin. 47: 488-495. https://doi.org/10.1093/abbs/gmv036
  15. Choi ES, Han SS, Cheong DE, Park M-Y, Kim J-S, Kim G-J. 2010. Generation of a fast maturating red fluorescent protein by a combined approach of elongation mutagenesis and functional salvage screening. Biochem. Biophys. Res. Commun. 391: 598-603. https://doi.org/10.1016/j.bbrc.2009.11.105