In-vitro Protein Production for Structure Determination with the Rapid Translation System (RTS)

摘要

The goal of the Berkeley Structural Genomics Center is to determine the structures of all proteins encoded in the genomes of Mycoplasma pneumoniae and Mycoplasma genitalium of structural homologs from other organisms. To achieve this goal, the Berkeley Structural Genomics Center is developing high-throughput methods for protein expression for use in X-ray and NMR structure determination. In collaboration with Roche Molecular Biochemicals, we are investigating the usefulness of the RTS for in-vitro protein production to generate target proteins in quantities suitable for structure determination. Very encouraging results have been obtained with the test protein phosphoserine phosphates from Methanococcus jannaschii. The structure of the phosphoserine phosphatase (PSP) from Methanococcus jannaschii has been determined previously[1]. In this study, PSP was produced in milligram quantities with the RTS, and the data collected by X-ray and NMR for structure determination of PSP were comparable with those of PSP produced in E. coli. Two labeling methods for NMR employing ~(15)N-glycine were compared.