Abstract
Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G< comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G (185 amino acids), we named this protein "protein Ge." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein Ge sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein Ge in E. coli. A dissociation constant (K d ) value of 9.4 nM for protein G<e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins. [Figure not available: see fulltext.]
Original language | English |
---|---|
Pages (from-to) | 482-492 |
Number of pages | 11 |
Journal | Journal of the American Society for Mass Spectrometry |
Volume | 26 |
Issue number | 3 |
DOIs | |
Publication status | Published - Mar 2015 |
Externally published | Yes |
Keywords
- Amino acid sequencing
- Bottom-up MS
- ESI-MS
- FTICR MS
- K value
- MALDI ToF MS
- PTM analysis
- Thermophoresis
- Top-down MS