Overexpression of functional hydrolase domain of rat liver 10-formyltetrahydrofolate dehydrogenase in Escherichia coli.
Rat liver 10-formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers. We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-formyltetrahydrofolate hydrolase, in Escherichia coli BL21 (DE3) cells using the pRSET expression vector. We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences. The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity. The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore). The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture. Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed in E. coli were similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells. The purified recombinant enzyme remained active for at least 4 weeks at 4 degreesC. These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme in E. coli cells and purified in one step by a simple chromatographic procedure.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, 37232, USA. krupensa@ctrvax.vanderbilt.edu
S A
SA
Krupenko
C
C
Wagner
D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000099295:Formyltetrahydrofolate Dehydrogenase
D017931:DNA Primers; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011994:Recombinant Proteins; D000099295:Formyltetrahydrofolate Dehydrogenase