Identification and quantification of N super( alpha )-acetylated Y. pestis fusion protein F1-V expressed in Escherichia coli using LCMS super(E)

• Published in: Journal of biotechnology 2007-05, Vol.130 (1), p.11-23
• Main Authors: Bariola, P A, Russell, BA, Monahan, S J, Stroop, S D
• Format: Article
• Language: English
• Subjects: Escherichia coli
• ISSN: 0168-1656
• DOI: 10.1016/j.jbiotec.2007.02.024

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 42-43Da 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+/-0.0231Da using a LockSpray(TM) 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 super(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N super( alpha )-acetylation. The average content of N super( 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 super( 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.