Identification and expression of the tig gene coding for trigger factor from psychrophilic bacteria with no information of genome sequence available.
Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis-trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic bacteria grow at a quite low temperature, between 4 and 15 degrees C, TF from such bacteria was investigated and compared with that of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival against cold treatment at 4 degrees C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity in a concentration dependent manner.
Lee
Kyunghee
K
Department of Chemistry, Sejong University, Seoul, 143 747, Korea. khlee@sejong.ac.kr
Choi
Hyojung
H
Im
Hana
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D003080:Cold Temperature; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D050296:Microbial Viability; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D019696:Peptidylprolyl Isomerase