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.

Physiochemical and functional characterization of antigen proteins eluted from aluminum hydroxide adjuvant.

We have characterized protein antigens after quantitative dissociation from aluminum hydroxide adjuvant. Bovine serum albumin (BSA) and a multi-antigen vaccine for Group A Streptococcus (GrAS Vaccine) were formulated on aluminum hydroxide, stored for > or =10 days then eluted with a 48-h treatment at 4 degrees C with 0.85% H(3)PO(4) plus 4M guanidine HCl (GnHCl). BSA is recovered from adjuvant at 92+/-2%. GrAS antigens are equally recovered from GrAS Vaccine (95+/-11% of total protein expected using multiple lots stored for up to 12 months). Recovery after elution is similar when determined by RP-HPLC, SEC-HPLC, UV absorbance, or Bradford methods. Eluted antigens are structurally and functionally intact as judged relative to both treated and untreated antigen controls by SDS-PAGE, RP-HPLC, SEC-HPLC, and after desalting by circular dichroism, bis-ANS binding, and antigenicity determined by ELISA. When formulated and stored for a few weeks, BSA has more dimer (31+/-5%) relative to the elution control (9% dimer) as detected by SEC-HPLC, suggesting that BSA microaggregation is promoted on aluminum. Antigens eluted from very aged GrAS Vaccine (>12 months) show marked changes by RP-HPLC. Structural changes in the antigens under elution conditions were evaluated using bis-ANS, a fluorescent probe of protein structure. Binding of bis-ANS increases fluorescence approximately 100-fold and is significantly diminished with increasing GnHCl concentrations indicating a progressive denaturing of the proteins. At 4M GnHCl (with or without 0.85% H(3)PO(4)) the GrAS antigens are fully denatured and BSA is partially denatured. Interestingly, the addition of 0.85% H(3)PO(4) increases bis-ANS binding on GrAS antigens and reduces the denaturing of GrAS antigens and BSA by chaotropes. Desalting or diluting the eluted antigens results in renaturing of the proteins as judged by bis-ANS fluorescence, circular dichroism and antigenicity testing. The elution method provides a novel approach for high recovery and characterization of GrAS Vaccine antigens and may be applicable to the study of many aluminum hydroxide-bound vaccines.