Rapid identification of specific mutations in the sequence of an enzyme variant produced by protein engineering using high-performance liquid chromatographic/fast atom bombardment mass spectrometric techniques.

Unknown, specific mutations in the sequence of an enzyme variant (a Bacillus subtilisin protease) produced by protein engineering were identified using High-performance Liquid Chromatographic/Fast Atom Bombardment Mass Spectrometric (HPLC/FAB MS) techniques. The variant and the highly homologous wild-type enzyme were treated with CNBr followed by tryptic digestion. The resulting peptides were analysed using HPLC/frit FAB MS. The peptides with molecular masses beyond the range of the HPLC/MS system under the chosen scanning conditions were collected using HPLC and subsequently analysed 'off-line' using static FAB MS. This procedure allowed the complete amino acid sequence determination of the variant protease using the known amino acid sequence of the wild-type enzyme as reference.

The gene for yeast ribosomal protein S31 contains an intron in the leader sequence.

Analysis of the primary structure of the gene for yeast ribosomal protein S31 revealed two unusual features. First, an intron of 312 nucleotides is located within the 5'-untranslated region. Second, the coding sequence for the known amino-terminal peptide of the protein starts 13 codons downstream of the ATG initiation codon, suggesting that S31 is synthesized as a precursor which undergoes post-translational processing to the mature protein. Primer extension analysis showed that transcription of the S31 gene starts at multiple sites. The 5'-flanking region of the gene contains several, previously described, conserved sequence elements that may play a role in the coordinate expression of yeast ribosomal protein genes.