Identification and quantification of N alpha-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS E.
N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.
Bariola
Pauline A
PA
GlaxoSmithKline Biologicals North America, 19204 North Creek Parkway, Bothell, WA 98011, USA.
Russell
Brett A
BA
Monahan
Steven J
SJ
Stroop
Steven D
SD
D000107:Acetylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D015010:Yersinia pestis / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C086725:caf1 protein, Yersinia pestis