Characterization of a mutant recombinant S100 protein using electrospray ionization mass spectrometry.
Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC). Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting. The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected. One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified. The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10. The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation. The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10. Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon. Another stop codon (TGA) located 27 base pairs downstream halts translation. The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass. Readthrough occurs in strains of E. coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.
Raftery
M J
MJ
Cytokine Research Unit, School of Pathology, University of New South Wales, Kensington, Australia.
Harrison
C A
CA
Geczy
C L
CL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D009418:S100 Proteins / Q000032:analysis* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D009418:S100 Proteins