Seamless GFP and GFP-Amylase Cloning in Gateway Shuttle Vector, Expression of the Recombinant Proteins in E. Coli and Bacillus Megaterium and Assessment of the GFP-Amylase Thermostability

Abstract

ABSTRACTA Gateway shuttle vector pMMC for gene cloning and expression in E. coli and Bacillus megaterium hosts was constructed using pMM1525. Together with the application of a PCR fusion/Gateway cloning procedure established earlier, the constructed vector makes possible efficient and very flexible seamless gene cloning in a sequence-independent manner A Gateway entry clone pXZ containing the XylR/pXylA' cassette of the XylR-xylose-dependent repressor gene and the pXylA'-promoter region of the xylose isomerase gene from the xylose operon of B. megaterium, was constructed. The XylR/pXylA '-mediated gene cloning in the constructed vector provides tight regulation of the gene expression in B. megaterium host cells upon xylose induction and constitutive expression in E.coli host cells. The efficiency of the seamless gene cloning was demonstrated through cloning in pMMC of the XylR/pXylA'-GFP and (XylR/pXylA'-GFP)-BLA expression cassettes, where GFP is the GFPuv variant of the green fluorescent protein gene and BLA is the Bacillus licheniformis α-amylase A gene. A mild and efficient thermolysis procedure for extraction of thermostable recombinant proteins expressed in E. coli was designed. It includes heating of lysozyme-treated E. coli cells at 50 °C and precipitation of the thermally denatured proteins. The comparative characterization of protein preps from E. coli cells expressing the GFP-BLA fusion protein and GFP alone revealed that GFP-BLA shows fluorescent ability with high thermal stability and has α-amylase activity similar to those of BLA proteins. The possible applications of the constructed pMMC Gateway shuttle vector and different protocols of the PCR fusion/Gateway cloning procedure for accomplishment of various gene cloning and expression tasks are discussed in details. The observed functionality and high thermal stability of the GFP-BLA fusion protein could serve to propose an integrated approach for characterization of the functionality and thermal stability of the GH13 and GH57 glycosyl hydrolase genes cloned from environmental DNA samples, fused with a GFP reporter gene and expressed in E. coli and/or B. megaterium hosts.