Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

Abstract

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.

A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency

“…For a quantitative measure of IMP expression, we employ a C-terminal fusion-tag of a green fluorescent protein (GFP) variant (Waldo et al, 1999) (Figure 1A) and measure whole-cell fluorescence by flow cytometry. Whole-cell fluorescence intensity of this fusion-tag has been validated in numerous previous studies to correlate strongly with the amount of folded IMP, rather than the total level of IMP translated (Fluman et al, 2014, Wang et al, 2011, Guglielmi et al, 2011, Geertsma et al, 2008, Drew et al, 2005); we further validate the expression levels measured from whole-cell fluorescence (Figure 1B) using in-gel fluorescence (Figure 1C and Figure S1, Pearson correlation coefficient, r = 0.914) and western blot analysis (Figure S1). With this approach, expression levels in E. coli are experimentally measured for TatC homologs from a variety of bacteria, including Aquifex aeolicus ( Aa ), Bordetella parapertussis ( Bp ), Campylobacter jejuni ( Cj ), Deinococcus radiodurans ( Dr ), Escherichia coli ( Ec ), Hydrogenivirga sp.…”

Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein

“…The OppC-GFP protein represented by the GFP band was in the folded form. The intensity of the non-denatured bands was closely related to the protein expression level, and was not affected by the sample preparation process (Wang et al, 2011). The fluorescent bands were proposed to represent the soluble portion of the target protein (Geertsma et al, 2008).…”

Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene

“…Adenovirus vectors are capable of infecting a broad range of mammalian cells for stable expression of recombinant proteins, which could assist in subsequent analyses (Ma et al, 2010;Wei et al, 2010). Tracing proteins, such as green fluorescent proteins (GFP), are widely used in the tracing of protein synthesis, position, and metabolism, without identifying the proteins using antibodies (Wang et al, 2011). X-GFP fusion proteins can be created artificially by genetic engineering, facilitating future biological research (Li et al, 1999;Wong et al, 2011;Yang et al, 2011).…”