TAILIEUCHUNG - Báo cáo khoa học: Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation

Apsychrotrophic bacteriumShewanellasp. strain SIB1 was grown at 4 and 20 C, and total soluble proteins extracted from the cells were analyzed by two-dimensional poly-acrylamide gel electrophoresis. Comparison ofthese pat-terns showed that the cellular content ofa protein with a molecular mass of28 kDa and an isoelectric point of four greatly increased at 4 C compared to that at 20 C. Determination ofthe N-terminal amino acid sequence, fol-lowed by the cloning and sequencing ofthe gene encoding this protein, revealed that this protein is a member ofthe FKBP family of proteins with an amino acid sequence identity of56% to Escherichia coliFKBP22. . | Eur. J. Biochem. 271 1372-1381 2004 FEBS 2004 doi Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation Yutaka Suzuki Mitsuru Haruki Kazufumi Takano Masaaki Morikawa and Shigenori Kanaya Department of Material and Life Science Graduate School of Engineering Osaka University Japan A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 C and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these att-terns showed that the cellular content ofa protein with a molecular mass of 28 kDa add an isoelectric point of four greatly increased at 4 C compared to that at 20 C. Determination of the N-termínal amino acid sequence. Iill-lowed by the cloning and sequencing of the gene encodíng this protein revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56 to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form purified and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures the protein exhibited the highest activity at 10 C with a kejtf Kn value of M-1 s-1. When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T1 refolding assay at 10 and 20 C the protein exhibited higher activity at 10 C with a kcat Km value of pM_1-s-1. These kc Km values are lower but comparable to those of E. ooli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation ofpsychrotrophic bacteria. Keywords psychrotrophic bacterium 2D-PAGE FKBP family protein PPIase cold-adaptation. Nascent polypeptides must be folded into their precise 3D structures to become functional proteins. As folding intermediates

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