title: Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.
abstract: BACKGROUND
  Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).
  
  
  RESULTS
  Overexpression of fusion proteins by IPTG induction 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, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). 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 overexpressing 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 fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction.
  
  
  CONCLUSIONS
  Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could be enhanced by maintaining the expression of σ32, a bacterial heat shock transcription factor, at higher levels during overproduction.
authors.affiliation: Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
authors.forename: Chung-Hsien
authors.initials: CH
authors.lastname: Cheng
authors.forename: Wen-Chien
authors.initials: WC
authors.lastname: Lee
mesh_terms: D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D015854:Up-Regulation
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D020543:Proteome; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase
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