A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide‐linked dimerisation of recombinant human insulin‐like growth factor‐1 secreted from yeast

FEBS Letters

Stephan, Christine¹  Chaudhuri, Bhabatosh¹ 

[1] Department of Biotechnology, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland

关键词: Protein-folding in vivo;    Insulin-like growth factor-1;    Intermolecular disulfide-bonded dimer;    Kex2 endoprotease;    Saccharomyces cerevisiae;    Yeast ER-retention signal HDEL;    αFL;    α-factor leader;    D;    aspartic acid;    DTT;    dithiothreitol;    E;    glutamic acid;    ELISA;    enzyme-linked immunoabsorbant assay;    ER;    endoplasmic reticulum;    H;    histidine;    HPLC;    high-performance liquid chromatography;    IGF1;    human insulin-like growth factor-1;    K;    lysine;    L;    leucine;    proαFL;    proregion of the αFL;    wt;    wild-type;   

DOI  :  10.1016/0014-5793(92)80585-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The majority of the recombinant human insulin-like growth factor-1 (IGF1) molecules, secreted from yeast using the prepro sequence of the prepro-α-factor, are not active monomers but inactive, disulfide-linked dimers. The prepro sequence of the prepro-α-factor, usually referred to as the α-factor leader (αFL), consists of a pre or signal sequence and a proregion. After signal sequence removal during translocation into the endoplasmic reticulum (ER) the proregion is still attached to IGF1 when it folds to acquire a tertiary structure. Mature IGF1 is released only in a late Golgi compartment by the membrane-bound endoprotease Kex2p. We find that co-expression of a novel ER-retained Kex-2p variant, soluble Kex2pHDEL, can prevent intermolecular disulfide bond formation between two IGF1 molecules, implying that the presence of the proregion during the folding of IGF1 in the ER could be a reason for disulfide-linked dimerisation. This result indicates that the proregion of the αFL may have a role in the folding of some heterologous proteins in yeast, and that the ER-retained Kex2p mutant could be used as a convenient tool to study the cellular function of the proregions present naturally in various eucaryotic precursor proteins.

【 授权许可】

Unknown