|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1994 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1993 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1992 : ¥800,000 (Direct Cost : ¥800,000)
We planned to develope the method for crystalization of membrane bound proteins. The method consists of two steps, firstly formation of two dimensional crystal of the protein utilizing the surface of water and then accumulation of it.
In order to perform this experiment, a large amount of the membrane protein is necessary. So we decided to purify E.coli membrane bound, detergent-resistant phospholipase A (DRPLA) since this enzyme had been well characterized for gene manipulation of DRPLA,generating an overproducing E.coli strain.
As the first step of purification, acetone precipitate of cell homogenate was solubilized with SDS and pretreated with Sepharose Q before Sepharose Q column chromatography. The crude preparation after the first column chromatography was indicated by SDS PAGE to still contain impurity proteins. As the next step, we performed an affinity column-chromatography of anti DRPLA polyclonal antibody because we failed in obtaining monoclonal antibody against DRPLA.
found that DRPLA solubilized by detergents such as triton x-100, tween 20, SDS was unable to bind to antibodies coupled to column resins, perhaps because DRPLA would be entrapped into micelles of detergents deeply. In stead of detergent solution, DRPLA was found in the partial mixture of aqueous solution with orgnic solvents to bind to antibodies, but DRPLA fractions eluted from the DRPLA-binding antibody columns still contained some impurities and, in addition, recovery yield of DRPLA was very low. Then, we attempted SDS polyacrylamide gel electrophoresis purification of DRPLA.A large scale of preparative electrophoresis was performed with the crude preparation and DRPLA band (29KD) was cut out from SDS PAGE gel. The apparatus was constructed for electrical elution of DRPLA from DRPLA band gel pieces. Some preliminary experiments indicated that almost pure DRPLA was eluted quite effectively from gel pieces and that this electrophoresis method is very useful for purification of other proteins. We shall continue this project with the obtained pure DRPLA. Less