Expression, Characterisation and Structural Homology Modelling of Recombinant Mercuric Reductase of Streptomyces sp. AS2

https://doi.org/10.22146/jtbb.88773

Anis Uswatun Khasanah(1), Wahyu Aristyaning Putri(2), Hanum Mukti Rahayu(3), Langkah Sembiring(4), Yekti Asih Purwestri(5*)

(1) Microbiology Laboratory, Department of Biology, Science Faculty, State Islamic University of Sultan Maulana Hasanuddin Banten. Jl. Jalan Jendral Sudirman No. 30 Panancangan Cipocok Jaya, Sumurpecung, Kec. Serang, Kota Serang, Banten 42118, Indonesia.
(2) Biotechnology Laboratory, Department of Tropical Biology, Universitas Gadjah Mada. Jl. Teknika Selatan, Sekip Utara, Bulaksumur Yogyakarta 55281, Indonesia.
(3) Program of Biology Education, Faculty of Teacher Training and Education, Universitas Muhammadiyah Pontianak. Jl. Ahmad Yani No. 111 Pontianak 78124, West Kalimantan, Indonesia.
(4) Microbiology Laboratory, Department of Tropical Biology, Universitas Gadjah Mada. Jl. Teknika Selatan, Sekip Utara, Bulaksumur Yogyakarta 55281, Indonesia.
(5) Biochemistry Laboratory, Department of Tropical Biology, Universitas Gadjah Mada. Jl. Teknika Selatan, Sekip Utara, Bulaksumur Yogyakarta 55281, Indonesia; Research Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
(*) Corresponding Author

Abstract


Mercury pollution poses a significant environmental challenge worldwide, prompting extensive efforts over the past two decades to combat its detrimental effects. Cloning merA from Streptomyces sp. AS2 (Accession numbers LC026157) into the expression vector pET-28c (+) marks a critical advancement in this field, necessitating further investigation into the expression and structural analysis of the resulting recombinant mercuric reductase protein. This study aimed to optimise the expression and characterise the structural MerA protein. The study involved the expression of merA from AS2 isolate in the host Escherichia coli BL21 and the measurement of mercuric reductase using SDS-PAGE. Induction of E. coli BL21 was optimized by adding IPTG concentration and incubation time. Purification of mercuric reductase was attempted using ammonium sulphate precipitation, dialysis, and column chromatography. Protein structural characterisation was conducted using computational modelling tools Swiss-Model and Phyre2. Expression of merA from AS2 isolate was successfully performed in E. coli BL21, with SDS-PAGE showing a dominant band in the 55-70 kDa range using IPTG concentration 1 and 1,2 mM and 18-hour incubation time. The specific activity of mercuric reductase was obtained at an enzyme concentration of 294.07 Unit/mg. Protein structural characterisation revealed homology with Lysinibacillus sphaericus (Swiss-Model) and similar folding to c5c1Yc, a known mercuric reductase from the same species using Phyre2. The successful expression of recombinant pET-28c (+)-MerA in E. coli BL21 offers new opportunities for bioremediation efforts targeting mercury contamination. 


Keywords


E. coli BL-21; gene cloning; mercuric reductase; protein expression

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References

Aprilyanto, V. & Sembiring, L., 2017. Bioinformatika, Innosain.

Bafana, A., Khan, F. & Suguna, A., 2017. Structural and functional characterization of mercuric reductase from Lysinibacillus sphaericus strain G1. Biometals, 30(5), pp.809–819. doi: 10.1007/s10534-017-0050-x.

Barkay, T. et al., 2010. A thermophilic bacterial origin and subsequent constraints by redox, light and salinity on the evolution of the microbial mercuric reductase. Environmental Microbiology, 12(11), pp.2904–2917. doi: 10.1111/j.1462-2920.2010.02260.x.

Bharagava, R., Chowdhary, P. & Saxena, G., 2017. Bioremediation: An Eco-Sustainable Green Technology: Its Applications and Limitations. In Environmental Pollutants and Their Bioremediation Approaches. Florida, USA: CRC Press, pp.1–22. doi: 10.1201/9781315173351-2.

Biasini, M. et al., 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research, 42(Web Server issue), pp.W252-258. doi: 10.1093/nar/gku340.

Centers for Disease Control and Prevention, 2004, ‘Elizabethkingia’, in Centers for Disease Control and Prevention, viewed 19 June 2024, from https://www.cdc.gov/elizabethkingia/about/index.html.

Cold, S.H.P., 2012, ‘SOC Medium for E. coli ‘, in Cold Spring Harbor Laboratory Press, viewed 13 June 2024, from https://cshprotocols.cshlp.org/content/2012/6/pdb.rec069732.short

Cuozzo, S.A. et al., 2018. Streptomyces sp. is a powerful biotechnological tool for the biodegradation of HCH isomers: biochemical and molecular basis. Critical Reviews in Biotechnology, 38(5), pp.719–728. doi: 10.1080/07388551.2017.1398133.

Dash, H.R. et al., 2017. Functional efficiency of MerA protein among diverse mercury resistant bacteria for efficient use in bioremediation of inorganic mercury. Biochimie, 142, pp.207–215. doi: 10.1016/j.biochi.2017.09.016.

Dereeper, A. et al., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research, 36(Web Server), pp.W465–W469. doi: 10.1093/nar/gkn180.

Green, M.R. & Sambrook, J., 2020. The inoue method for preparation and transformation of competent Escherichia coli: Ultracompetent cells. Cold Spring Harbor Protocols, 2020(6), 101196. doi: 10.1101/pdb.prot101196.

Jones, D.T., Taylor, W.R. & Thornton, J.M., 1992. The rapid generation of mutation data matrices from protein sequences. Computer applications in the biosciences: CABIOS, 8(3), pp.275–282. doi: 10.1093/bioinformatics/8.3.275.

Kelley, L.A. et al., 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nature Protocols, 10(6), pp.845–858. doi: 10.1038/nprot.2015.053.

Mello, I.S. et al., 2020. Endophytic bacteria stimulate mercury phytoremediation by modulating its bioaccumulation and volatilization. Ecotoxicology and Environmental Safety, 202, 110818. doi: 10.1016/j.ecoenv.2020.110818.

Ogunseitan, O.A., 1998. Protein method for investigating mercuric reductase gene expression in aquatic environments. Applied and Environmental Microbiology, 64(2), pp.695–702. doi: 10.1128/AEM.64.2.695-702.1998.

Paul, T. et al., 2020. A comprehensive review on recent trends in production , purification , and applications of prodigiosin. Biomass Conversion and Biorefinery, 12, pp.1409-1431. doi: 10.1007/s13399-020-00928-2.

Putri, W.A. et al., 2021. Identification of mercury‐resistant Streptomyces isolated from Cyperus rotundus L. rhizosphere and molecular cloning of mercury (II) reductase gene. Indonesian Journal of Biotechnology, 26(4), pp.206-213. doi: 10.22146/ijbiotech.65989.

Rahayu, H.M. et al., 2021. Indigeneous Streptomyces spp. isolated from Cyperus rotundus rhizosphere indicate high mercuric reductase activity as a potential bioremediation agent. Biodiversitas Journal of Biological Diversity, 22(3), pp.1519-1526. doi: 10.13057/biodiv/d220357.

Sambrook, J. & Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

Sezonov, G., Joseleau-Petit, D. & D’Ari, R., 2007. Escherichia coli physiology in Luria-Bertani Broth. Journal of Bacteriology, 189(23), pp.8746-8749. doi: 10.1128/JB.01368-07.

Singh, S. & Kumar, V., 2020. Mercury detoxification by absorption, mercuric ion reductase, and exopolysaccharides: a comprehensive study. Environmental Science and Pollution Research, 27(22), pp.27181–27201. doi: 10.1007/s11356-019-04974-w.

Tamura, K., Stecher, G. & Kumar, S., 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38(7), pp.3022–3027. doi: 10.1093/molbev/msab120.

Terefe-Ayana, D. et al., 2012. Evolution of the Rdr1 TNL-cluster in roses and other Rosaceous species. BMC Genomics, 13, 409. doi: 10.1186/1471-2164-13-409.

Zeroual, Y. et al., 2003. Purification and characterization of cytosolic mercuric reductase from Klebsiella pneumoniae. Annals of Microbiology, 53, pp.149–160.

Zeyaullah, M et al., 2010. Molecular cloning and expression of bacterial mercuric reductase gene. African Journal of Biotechnology, 9(25), pp.3714-3718.



DOI: https://doi.org/10.22146/jtbb.88773

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