FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli.
The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.
Ideno
Akira
A
Marine Biotechnology Institute Co., Ltd., Kamaishi Laboratories, 3-75-1 Heita, Kamaishi, Iwate 026-000, Japan. akira.ideno@kamaishi.mbio.co.jp
Furutani
Masahiro
M
Iba
Yoshitaka
Y
Kurosawa
Yoshikazu
Y
Maruyama
Tadashi
T
D000818:Animals; D015153:Blotting, Western; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D006358:Hot Temperature; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D009113:Muramidase / Q000276:immunology; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry* / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D013884:Thiosulfate Sulfurtransferase; D009113:Muramidase; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase