Over-expression in Escherichia coli, functional characterization and refolding of rat dimethylglycine dehydrogenase

• Published in: Protein expression and purification 2004-10, Vol.37 (2), p.434-442
• Main Authors: Brizio, Carmen, Brandsch, Roderich, Bufano, Daniela, Pochini, Lorena, Indiveri, Cesare, Barile, Maria
• Format: Article
• Language: English
• Subjects: Animals; Biochemistry - methods; Blotting, Western; Dimethylglycine Dehydrogenase; Electrophoresis, Polyacrylamide Gel; Escherichia coli - enzymology; Escherichia coli - metabolism; FAD; Flavinylation; Flavoprotein; Hydrogen-Ion Concentration; Liver - enzymology; Mitochondria; Mitochondrial Proteins; Nickel - chemistry; Oxidoreductases, N-Demethylating - chemistry; Plasmids - metabolism; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Proteins - chemistry; Refolding; Sarcosine - analogs & derivatives; Sarcosine - chemistry
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.06.011

Dimethylglycine dehydrogenase (Me 2GlyDH) is a mitochondrial enzyme that catalyzes the oxidative demethylation of dimethylglycine to sarcosine. The enzyme requires flavin adenine dinucleotide (FAD), which is covalently bound to the apoprotein via a histidyl(N3)-(8α)FAD linkage. In the present study, the mature form of rat Me 2GlyDH has been over-expressed in Escherichia coli as an N-terminally 6-His-tagged fusion protein. The over-expressed protein distributed almost equally between the soluble and insoluble (inclusion bodies) cell fraction. By applying the soluble cell lysate to a nickel-chelating column, two fractions were eluted, both containing a nearly homogeneous protein with a molecular mass of 93 kDa, on SDS–PAGE. The first protein fraction was identified by Western blotting analysis as the covalently flavinylated Me 2GlyDH. It showed optical properties and specific activity (240 nmol/min/mg protein) similar to those of the native holoenzyme. The second fraction was identified as an underflavinylated (apo-) form of Me 2GlyDH, with a 70% lower specific activity. The recombinant holoenzyme exhibited optimal activity at pH 8.5, an activation energy of about 80 kJ/mol, and two K M values for N, N-dimethylglycine ( K M1=0.05 mM and K M2=9.4 mM), as described for the native holoenzyme. Starting from the inclusion bodies, the unfolded flavinylated enzyme was solubilized by SDS treatment and refolded by an 80-fold dilution step, with a reactivation yield of 50–60%.