PCR cloning of the resuscitation-promoting factor (Rpf) gene from Micrococcus luteus, sequencing and expression in Escherichia coli.
A polymerase chain reaction (PCR) cloning procedure was developed for the resuscitation-promoting factor (Rpf) gene of Micrococcus luteus using strains NCIMB 13267, JCM 1464T, JCM 3347, and JCM 3348. A PCR product of the Rpf gene fragment was ligated into a cloning vector pBluescript II KS (+) with the restriction endonucleases Eco RI and Bam HI. The ligation mixture was used to transform Escherichia coli DH5alpha. The DNA sequence of the Rpf gene cloned from strain JCM 1464T was 84% homologous with that of NCIMB 13267, and from strains JCM 3347 and JCM 3348 it was 100% and 86% homologous, respectively. Recombinant Rpf proteins of M. luteus NCIMB 13267 and JCM 1464T after expression in E. coli BL21 harbouring the pET-19b-Rpf plasmid, and after purification, were approximately 16 kD for both strains.
Laboratory of Molecular Biology, Graduate School of Environmental Health Sciences, Azabu University, Sagamihara, Japan.
M
M
Matsuda
M
M
Togo
S
S
Kagawa
J E
JE
Moore
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016982:Micrococcus luteus / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D016207:Cytokines; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C113704:resuscitation-promoting factor, bacteria