Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis-trans isomerase (N-PPlase) and double-labeled ( C-13- and N-15-) protein (DL-PPlase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPlase and four bands for DL-PP lase. Since the N-terminal amino acid Analysis, Central residues of all ban ds were the same, we characterized these bands using the peptide mapping me thod and amino acid composition analysis. Peptide mapping of the proteins s eemed to be almost identical but they could not reflect the whole amino aci d sequences of the protein. The bands on the polyvinylidene difluoride (PVD F) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (S DS-PAGE), were hydrolyzed and their amino acid composition was analyzed usi ng a highly sensitive 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (Sigma(T%-E%)(2)) for similarity of proteins was calculated by summation of the square difference between the theoretical (T %) and th e experimental (E %) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins wer e 16 812 and 17 694 by electrospray ionization (ESI)-mass spectrometry. How ever, the purified proteins also contained minor compounds with M-r of 3 72 1 for N-PPlase and 5 285 for DL-PPlase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of t he amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for char acterization of a recombinant protein.