[Gene expression, purification and functional analysis of LiPrmA].
Leptospira interrogans (L. interrogans) genomic DNA was used as template to amplify the full-length gene for ribosomal protein L11 methyltransferase (liPrmA) by PCR. The pET22b-/liprmA expression plasmid was successfully constructed in Escherichia coli (E.coli.) strain TOP10 and confirmed by restriction enzyme digest and sequencing. Through optimizing expression of the recombinant liPrmA-6xHis fusion protein in expression host E. coli. BL21, the yield of soluble target protein reached 40 mg (liter culture)-1. The LiPrmA was purified to apparent homogeneity in a single step using Ni-NTA His Bind chromatography. Amino acid homologous analysis showed that liPrmA shared significant identity with other prokaryotic PrmA and eukaryotic putative PrmA in the catalytic region including AdoMet binding domain. Methylation activity experiments showed purified liPrmA was able to catalyze the ribosomal protein L11 of L. interrogans methylated under the presence of S-adenosyl-methionine (AdoMet).
Sun
Yan-Ju
YJ
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
Zhou
Zhong-Wei
ZW
Yao
Jian-Xiu
JX
Sun
Ti-Chang
TC
Xu
Yan-Yan
YY
Wen
Ya
Y
Bao
Shi-Lai
SL
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007921:Leptospira interrogans / Q000201:enzymology* / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; C084165:L11 methyltransferase; D008780:Methyltransferases