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-PPIase) and double-labeled (13C- and 15N-) protein (DL-PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPIase and four bands for DL-PPIase. Since the N-terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), were hydrolyzed and their amino acid composition was analyzed using 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 the 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 were 16812 and 17694 by electrospray ionization (ESI)-mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3721 for N-PPIase and 5285 for DL-PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the 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 characterization of a recombinant protein.
Murayama
K
K
Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Tokyo, Japan. murayama@med.juntendo.ac.jp
Shindo
N
N
Suzuki
R
R
Kawakami
M
M
Mineki
R
R
Taka
H
H
Kazuno
S
S
Nagata
K
K
Maruyama
T
T
Tanokura
M
M
D000596:Amino Acids / Q000032:analysis; D019641:DNA, Archaeal; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D013058:Mass Spectrometry; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D010449:Peptide Mapping; D019696:Peptidylprolyl Isomerase / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D012967:Sodium Dodecyl Sulfate
D000596:Amino Acids; D019641:DNA, Archaeal; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D019696:Peptidylprolyl Isomerase