14 化学法と発現法を組み合わせた糖タンパク質エリスロポエチン誘導体の合成(口頭発表の部)

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概要

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• To investigate the function of oligosaccharide on protein, we have examined synthesis of homogeneous glycoproteins by use of combined chemical and E. coli expression system. In our strategy, a homogeneous glycopeptide thioester is synthesized by chemical method, while normal peptide chain is prepared by E. coli expression system. Then coupling between this glycopeptide thioester and a large polypeptide chain prepared by E. coli expression system, can be performed by Native Chemical Ligation (NCL). In this study, we selected erythropoietin (EPO) analogue, which is consist of 166-amino acid residues and display two N-linked complex type disialyloligosaccharide chains at the 24 and 30 positions on the polypeptide chain. EPO analogue was divided into two segments, EPO segment (1-32) 5 and EPO segment (33-166) 7. In order to synthesize glycopeptides thioester segment 5 having two cysteine residues at the 24 and 30 positions where would be incorporated with oligosaccharides, protected peptide chain (1-32) was prepared by Fmoc SPPS and then its C-terminal carboxylic acid was converted into ethyl-thioester to afford peptide thioester 3. A complex-type disialyloligosaccharyl-bromoacetamide was employed to modify the two free cysteine residues. This coupling reaction afforded the desired neoglycopeptide thioester 5 in good yield. The EPO polypeptide chain, comprising residues 33-166 (7) was purified from E. coli as a polyhistidine-tagged fusion protein with a methionine residue inserted between the tag and the EPO segment (33-166) 6. After isolation of the fusion protein 6 by nickel-affinity column, Histag-methionine segment was removed by CNBr treatment to afford desired EPO segment having cysteine residue at the N-terminus. Finally, we examined coupling reaction between these two segments by NCL and this reaction afforded whole EPO analogue in moderate yield. Finally, we examined folding experiment in order to have bioactive EPO analogue. In this presentation, we would like to discuss synthesis of EPO analogue 10, its folding experiments and bioactivity.

• 2008-09-01

著者

• 梶原 康宏

  横浜市立大学大学院国際総合科学研究科

• Macmillan Derek

  University College London

• 平野 桐子

  横浜市立大学・院

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