An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl-prolyl cis/trans isomerase activity

FEBS Letters
G StollerG Fischer

Abstract

The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl-prolyl cis/trans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatically active fragment could be isolated after proteolysis by subtilisin. The resulting polypeptide was analysed by N-terminal sequencing and MALDI-TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg-145 to Glu-251. The nucleotide sequence encoding the amino acid residues Met-140 to Ala-250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His-tagged polypeptide was isolated on an Ni2+-NTA column. Subsequent digestion with subtilisin and anion-exchange chromatography yielded a TF fragment encompassing amino acids Gln-148 to Thr-249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant kcat/Km of 1.3 microM(-1) s(-1) could be demonstrated when using Suc-Ala-Phe-Pro-Phe-NH-Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity o...Continue Reading

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Citations

Jul 1, 2010·World Journal of Microbiology & Biotechnology·Qi-Ming ZhouJun-Mei Zhou
Feb 1, 2003·Journal of Molecular Biology·Raimund MaierFranz-Xaver Schmid
Feb 13, 2003·Journal of Molecular Biology·Gregor BlahaKnud H Nierhaus
Sep 9, 2004·Proceedings of the National Academy of Sciences of the United States of America·Anthony V LudlamZhaohui Xu
Nov 29, 2001·Proceedings of the National Academy of Sciences of the United States of America·H PatzeltE Deuerling
May 13, 1997·Proceedings of the National Academy of Sciences of the United States of America·O Kandror, A L Goldberg
Feb 27, 2003·Molecular Microbiology·Elke DeuerlingBernd Bukau
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Aug 28, 1998·FEBS Letters·C SchieneG Fischer
Sep 1, 2005·Yeast·Trevor J Pemberton, John E Kay
Aug 24, 2006·The Journal of Biological Chemistry·Frieder MerzElke Deuerling
Nov 7, 2003·The Journal of Biological Chemistry·Ryoji SunoMasasuke Yoshida
Feb 6, 2002·Chembiochem : a European Journal of Chemical Biology·F Bordusa
Dec 11, 2007·Biochemistry·Jiang-Bi Xie, Jun-Mei Zhou

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