In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence.
Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.
Yokoyama
Kei-ichi
K
Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki, 210-8681, Kanagawa, Japan. keiichia_yokoyama@ajinimoto.com
Kunio
Ono
O
Ohtsuka
Tomoko
T
Nakamura
Nami
N
Seguro
Katsuya
K
Ejima
Daisuke
D
D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*; D014508:Urea / Q000737:chemistry*
D014508:Urea; D011503:Transglutaminases