| Budget Amount *help |
¥4,120,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥420,000)
Fiscal Year 2007: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2006: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
To investigate 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 and then coupling between this glycopeptide thioester and a large polypeptide chain prepared by E. coli expression system, is performed by Native Chemical Ligation (NCL). In this study, we finally selected erythropoietin (EPO) analogue, which 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) and EPO segment (33-166). In order to synthesize glycopeptides thioester segment 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. A complex-type disialyloligosaccharyl-bromoacetamide was employed to modify the two free cysteine residues to obtain the neoglycopeptide thioester. This coupling reaction afforded the desired neoglycopeptide thioester (EPO:1-32) in good yield. The EPO polypeptide chain, comprising residues 33-166 was purified from E. coli as a polyhistidine-tagged fusion protein with a methionine residue inserted between the tag and the EPO sequence. After isolation of the fusion protein through 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. After folding experiment, we obtained desired EPO analogue by use of combined chemical and E. coil expression system.
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