Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive Escherichia coli protein, SlyD.
The proteome profile of Escherichia coli BL21(DE3) generated in response to heat shock stress was analyzed by two-dimensional electrophoresis (2-DE), wherein we identified a FKBP-type peptidyl-prolyl cis-trans isomerse (PPIases), SlyD, as a stress-responsive (i.e. aggregation-resistant) protein. Even under an imposed severe stress condition where 29 out of 858 soluble proteins were totally eliminated and the synthesis levels of 171 proteins decreased over 5-fold, a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition. As a fusion partner, as well as solubility enhancer, SlyD facilitated folding and significantly increased the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm. SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions and the formation of inclusion bodies, most likely as a result of intrinsic folding efficiencies and/or chaperone-like activities. SlyD was also shown to be suitable for the production of a biologically active fusion mutant of Pseudomonas putida cutinase that is of considerable biotechnological and commercial interest.
Han
Kyung-Yeon
KY
Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1, Sungbuk-Ku, Seoul 136-713, South Korea.
Song
Jong-Am
JA
Ahn
Keum-Young
KY
Park
Jin-Seung
JS
Seo
Hyuk-Seong
HS
Lee
Jeewon
J
D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D018869:Heat-Shock Response; D006358:Hot Temperature; D050296:Microbial Viability; D009154:Mutation / Q000235:genetics; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D029968:Escherichia coli Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; C085606:SlyD protein, E coli; D019696:Peptidylprolyl Isomerase