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

Stoller, Gerlind¹  Tradler, Thomas¹  Rücknagel, Karl P.¹  Rahfeld, Jens-U.¹  Fischer, Gunter¹ 

[1] Max-Planck-Gesellschaft, Arbeitsgruppe ‘Enzymologie der Peptidbindung’, Kurt-Mothes-Str. 3, D-06120 Halle/S, Germany

关键词: Limited proteolysis;    Peptidyl-prolyl cis/trans isomerase;    Trigger factor;    E. coli;    TF;    trigger factor;    PPIase;    peptidyl-prolyl cis/trans isomerase;    PMSF;    phenylmethylsulfonyl fluoride;    BSA;    bovine serum albumin;    -NH-Np;    4-nitroanilide;    Suc;    succinyl;    FKBP;    FK506-binding protein;   

DOI  :  10.1016/0014-5793(96)00282-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 k _cat /K _m of 1.3 μM⁻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 of the fragment was not inhibited by the peptidomacrolide FK506.

【 授权许可】

Unknown