Protein solubility and differential proteomic profiling of recombinant Escherichia coli overexpressing double-tagged fusion proteins.
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.
Cheng
Chung-Hsien
CH
Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Chiayi 621, Taiwan.
Lee
Wen-Chien
WC
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
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
Electrophoresis. 2000 Nov;21(17):3666-72
11271485
Appl Environ Microbiol. 2003 Oct;69(10):5772-81
14532024
Biotechnol Bioeng. 1999 Nov 20;65(4):382-8
10506413
Trends Biotechnol. 2006 Apr;24(4):179-85
16503059
Microb Cell Fact. 2006 Nov 17;5:34
17112383
J Struct Biol. 2001 Aug;135(2):84-93
11580258
J Bacteriol. 2005 Jul;187(13):4362-71
15968045
Metab Eng. 2000 Jul;2(3):201-9
11056062
Proteomics. 2009 Aug;9(16):4017-28
19658099
Biotechnol Bioeng. 2000 Oct 20;70(2):217-24
10972933
PLoS One. 2007 May 16;2(5):e447
17505547
Microbiol Mol Biol Rev. 2006 Jun;70(2):362-439
16760308
BMC Biotechnol. 2009 Jul 09;9:63
19586552
Appl Microbiol Biotechnol. 2003 Apr;61(2):163-78
12655459
Biotechnol Bioeng. 2003 Dec 30;84(7):801-14
14708121
Adv Biochem Eng Biotechnol. 2004;89:93-142
15217157
Nat Protoc. 2007;2(8):1896-906
17703201
Nucleic Acids Res. 1999 Oct 1;27(19):3821-35
10481021
Nucleic Acids Res. 1981 Jul 10;9(13):3105-17
7024910
Biotechnol Bioeng. 2004 Nov 20;88(4):426-36
15382106
Proteomics. 2003 Jul;3(7):1365-73
12872237
Biotechnol Lett. 2003 Sep;25(17):1385-95
14514038
J Biotechnol. 2005 Jan 26;115(2):113-28
15607230
Nat Protoc. 2006;1(6):2856-60
17406544
BMC Microbiol. 2006 Mar 10;6:27
16529647
Microb Cell Fact. 2005 Dec 13;4:34
16351710
Genes Dev. 1989 Aug;3(8):1226-32
2676724
J Bacteriol. 1992 Nov;174(21):6938-47
1356969
J Mol Biol. 2007 Nov 16;374(1):195-205
17920630
Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):8128-32
11416161
Appl Environ Microbiol. 2005 Apr;71(4):1717-28
15811994
Biotechnol Prog. 2005 Sep-Oct;21(5):1401-11
16209543
Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5808-13
8650174
Genes Dev. 2004 Nov 15;18(22):2812-21
15545634
Proteome Sci. 2005 May 31;3:4
15927046
BMC Biotechnol. 2007 Jun 12;7:32
17565681