Proteomic study on recombinant Escherichia coli overexpressing double-tagged fusion proteins and the improvement of protein solubility

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The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing two sequential sialic acid-producing enzymes, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both overexpressed with glutathione S-transferase (GST) and polyionic peptide (5D or 5R) tags. Overexpression of recombinant proteins by IPTG induction for three hours caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB and was coupled to the production of NADPH for cell biosynthesis, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). The expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring strains in response to over-production of recombinant proteins. In comparison with overexpressing aggregation-prone proteins, overexpression of soluble GST led to a lower expression of the disaggregation chaperone, ClpB and other chaperones. Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells over-expressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble faction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells over-expressing recombinant GST-GlcNAc 2-epimerase-5D, which has a higher probability of solubility according to bioinformatic predictions, the expression of σ32 was maintained at a higher level following induction. A potential strategy to promote the solubility of over-expressed recombinant proteins could be to maintain the enhanced expression of σ32. Therefore, the gene of sigma factor 32 (rpoH) from E. coli BL21 was cloned into vector pBAD33 and the resultant recombinant plasmid named pBAD33-rpoH was sent into E. coli harboring pGEX-2TK-nana-5R. Co-induction by IPTG and L-arabinose made cellular RpoH increase to a level about three times the basal expression in the pGEX-2TK-nana-5R-expressing cell, and meanwhile the solubility and soluble yield of GST-Neu5Ac aldolase-5R was improved by 34% and 62%.