Disulphide bridge formation of proinsulin fusion proteins during secretion in Streptomyces.
To study disulphide bridge formation by Streptomyces lividans TK 24 in secreted single chain precursors of insulin a fusion protein (PTF 1) was investigated consisting of monkey proinsulin and the aminoterminal sequence Asp1 to Gly43 of the alpha-amylase inhibitor tendamistat from Streptomyces tendae. The purified soluble protein PTF 1 has a molecular mass of 14.4 kDa. The primary structure was elucidated after digestion with lysyl endopeptidase and fragment analysis. In this system, disulphide bond formation occurs in a way that the first cysteine in proinsulin is linked to the next following cysteine in the amino-acid chain resulting in a non-natural folding of the insulin part of the fusion protein. Re-folding of PTF 1 by reduction and re-oxidation followed by proteolytic digestions led to insulins which are identical to authentic material. The ease of correct disulphide bond formation in solution and incorrect processing during secretion suggests involvement of yet unknown factors leading to an unfavourable folding of proinsulin.
Vértesy
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Hoechst AG, Frankfurt/Main.
Tripier
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D
Koller
K P
KP
Riess
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G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism*; D010450:Endopeptidases; D015870:Gene Expression; D008252:Macaca fascicularis; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011384:Proinsulin / Q000096:biosynthesis*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012697:Serine Endopeptidases / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism*
D004220:Disulfides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011384:Proinsulin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; C030526:tendamistate