|Budget Amount *help
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1991: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
We focus on how the sugar chain of protein effects on its protein refolding. This study may give a primitive information for choosing which is suitable, animal cell or bacteria, for the production of glycoprotein by using gene engineering. We also focus on the analyses of the relationship between the formation of disulfide bonds and that of three dimensional structure of proteins.
Ovalbumin (OVA) is one of the most suitable proteins for this purpose, because it is a glycoprotein, it is easy to purify, and that lots of information about the structure are available. In this project, we especially investigated the analysis of refolding of OVA, preparation of monoclonal antibody to the denatured OVA, and the effects of protein disulfide-isomerase (PDI) on the protein refolding.
Hirose et al. found that the native OVA can be refolded from its denatured form without disulfide bond formation, and its native disulfide bond can be formed later. But we investigated the system which could analyze both the protein refolding and the disulfide formation to study on the effect of PDI. We used "two step alkylation method", "trypsin degradation analysis", and the analysis of disulfide bond pairing by HPLC, for the structural analyses of OVA. First, we investigated the effect of PDI on OVA refolding, but we haven't found the promoting effect of PDI on its refolding yet. Second, we tried to prepare the monoclonal antibodies to the denatured OVA. But, we haven't got any specific antibody to its denatured from or to its domain of OVA. These results suggest that the refolding of OVA proceed too promptly, and each domain is in the native structure even if OVA itself is denatured. We don't have any results that the sugar chain of OVA influences its refolding so for. One reason may be that the sugar chain of OVA is too small.
We will focus on the interaction between OVA and PDI, and also focus on the refolding of other proteins which have both sugar chains and disulfide bonds.