The rate‐limiting steps for the folding of an antibody scFv fragment

FEBS Letters

Jäger, Marcus¹  Plückthun, Andreas¹ 

[1] Biochemisches Institut der Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland

关键词: Protein folding;    Proline cis/trans isomerization;    Folding intermediate;    scFv fragment;    Antibody engineering;   

DOI  :  10.1016/S0014-5793(97)01350-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from the phosphorylcholine binding antibody McPC603, was investigated. Both prolyl-peptide bonds which are cis in the native state affect the refolding kinetics of long-term denatured protein. The rate-limiting step is the trans→cis isomerization at the ProL95-peptide bond, which is catalyzed by peptidyl-prolyl-cis/trans-isomerase (PPIase), and is the prerequisite for correct V_H/V_L domain association. Refolding of short-term denatured protein resulted in complex refolding kinetics, too. This kinetic heterogeneity could be ascribed to cis→trans re-isomerization at the ProL95-peptide bond to the wrong conformation in a folding intermediate. PPIase was shown to increase the fraction of slowly folding species, thereby competing with the fast folding of short-term denatured scFv, having native proline conformations. A trapped intermediate is rapidly populated, and the return from this state becomes rate-limiting.

【 授权许可】

Unknown