Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase.
The Rickettsia prowazekii ATP/ADP translocase (Tlc) is the first member of a new family of ATP/ADP exchangers that includes both prokaryotic and eukaryotic proteins. We optimized the codon usage for expression of tlc in Escherichia coli by means of gene synthesis, expressed the synthetic gene in E. coli, and purified a modified Tlc that contained a C-terminal tag of 10 consecutive histidine residues by immobilized metal affinity chromatography. Although codon usage in R. prowazekii is very different from E. coli, the optimization of the codon usage by itself was insufficient to improve expression. However, the change of the cloning vector from pET11a to pT7-5 led to a 3-10-fold increase in the specific ATP transport rate by cells expressing the synthetic construct. The authenticity of the purified protein was confirmed by N-terminal amino acid sequencing and a matrix assisted laser desorption/ionization mass spectrometry.
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
M F
MF
Alexeyev
H H
HH
Winkler
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D000226:Mitochondrial ADP, ATP Translocases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012283:Rickettsia prowazekii / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000226:Mitochondrial ADP, ATP Translocases