A cyclophilin from Griffithsia japonica has thermoprotective activity and is affected by CsA.
Members of the multifunctional Cyp family have been isolated from a wide range of organisms. However, few functional studies have been performed on the role of these proteins as chaperones in red alga. For studying the function of cDNA GjCyp-1 isolated from the red alga (Griffithsia japonica), we expressed and purified a recombinant GjCyp-1 containing a hexahistidine tag at the amino-terminus in Escherichia coli. An expressed fusion protein, H6GjCyp-1 maintained the stability of E. coli proteins up to 50 degrees C. For a functional bioassay for recombinant H6GjCyp-1, the viability of E. coli cells overexpressing H6GjCyp-1 was compared with that of cells not expressing H6GjCyp-1 at 50 degrees C. After high temperature treatment for 1 h, E. coli overexpressing H6GjCyp-1 survived about three times longer than E. coli lacking H6GjCyp-1. Measurement of the light scattering of luciferase (luc) showed that GjCyp-1 prevents the aggregation of luc during mild heat stress and that the thermoprotective activity of GjCyp-1 is blocked by cyclosporin A (CsA), an inhibitor of Cyps. Furthermore, the Cyp-CsA complex inhibited the growth of E. coli under normal conditions. The results of the GjCyp-1 bioassays as well as in vitro studies strongly suggest that Cyp confers thermotolerance to E. coli.
Cho
Eun Kyung
EK
Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea. choeun@snu.ac.kr
Lee
Yoo Kyung
YK
Hong
Choo Bong
CB
D003001:Cloning, Molecular; D021983:Cyclophilins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D008156:Luciferases / Q000502:physiology; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000461:Rhodophyta / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D013997:Time Factors
C471213:His-His-His-His-His-His; D018832:Molecular Chaperones; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D016572:Cyclosporine; D008156:Luciferases; D021983:Cyclophilins