Recombinant protein overexpression, purification and In silico analysis of Maf protein

Abstract

Unlike the days when large amounts of animal and plant tissue or tissue fluid were needed to obtain a small amount of protein, recombinant protein technology has revolutionized as a go to approach to obtain purified proteins. With the development of new protocols, engineered bacterial strains, and plasmids, the recombinant protein expression has become efficient and widely available, allowing cost-effective production of specific proteins for scientific and applied purposes including industry, diagnostics and therapeutic applications. Though in theory, the steps appear simple, challenges can be at any level in practice impeding successful protein expression. Therefore, understanding the importance of contributions to the field, our objective was to overexpress recombinant maf gene, purify the protein, and In silico analysis. Multicopy associated filamentation (maf) gene of Caldimonas manganoxidans MS1, moderately thermophilic bacteria identified and characterized from MahaOya hot water springs in Ampara district, Sri Lanka and successfully cloned in pET28a (+)/E. coli BL21(DE3) system in a previous study was used for the overexpression of recombinant Maf protein. Glycerol stocks of the expression host E. coli BL21 (DE3) pLysS cells harboring the recombinant maf gene were acquired from -800C, revived by growth in LB agar plates and confirmed with polymerase chain reaction with primers specific to the maf gene. Bioinformatics analysis carried out using NCBI tools confirmed the identity of maf gene PCR product sequence with a 99% sequence similarity with the maf gene of Caldimonas manganoxidans (NZ_KB905929.1). The overexpression of the recombinant Maf protein was induced at the mid exponential phase of growth by the addition of isopropyl-ß-D thiogalactopyranoside (IPTG) to a final concentration of 1mM. Overexpressed recombinant Polyhistidine tagged Maf protein was purified using MagneHis TM Protein purification system with paramagnetic precharged Nickel particles. The protein structure was predicted using Swiss Model software of ExPASy while errat procheck and Ramachandran plot validated the structure, revealing approximately 94.4% of the amino acids in the favored region. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) with protein visualization by Coomassie Brilliant Blue R-250 staining indicated a single band at around 22kDa complying with the molecular weight expected by bioinformatics analysis. Therefore, in the present study recombinant Maf protein was successfully overexpressed, purified and verified with SDS PAGE analysis and bioinformatics analysis, providing prospects for scale up and further functional analysis of the recombinant protein