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

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