GroE-dependent expression and purification of pig heart mitochondrial citrate synthase in Escherichia coli.
Citrate synthase (CS) is a dimeric, mitochondrial protein, composed of two identical subunits (M(r) 48969 each). The nuclear-encoded alpha-helical protein is imported into mitochondria post-translationally where it catalyses the first step of the citric cycle. Furthermore, the pathway of thermal unfolding as well as the folding pathway was studied extensively, making CS a well-suited substrate protein for studying chaperone function. In chaperone research the quality of the substrate proteins is essential to guaranty the reproducibility of the results. In this context, we here describe the GroE-enhanced recombinant expression and purification of CS. CS was expressed in E. coli by using an arabinose regulated T7 promotor. Under standard expression conditions only insoluble, inactive CS was detected. Interestingly, the expression of soluble and active CS was possible when GroEL/GroES was co-expressed. Furthermore, a shift to lower expression temperatures increased the amount of soluble, active CS. We describe for the first time, the purification of CS in soluble and active form by following a CiPP strategy (capture, intermediate purification, polishing). After the initial capturing step on DEAE-Sephacel the protein was further purified on a Q-Sepharose column. After these two steps of anion-exchange chromatography a final size-exclusion chromatography step on a Superdex 75-pg column yields CS with a purity over 99%. Using this expression and purification strategy 1 mg CS per g E. coli wet weight were purified.
Haslbeck
Martin
M
Institut für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747, Garching, Germany. martin.haslbeck@ch.tum.de
Schuster
Ioana
I
Grallert
Holger
H
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D018833:Chaperonins; D002852:Chromatography, Ion Exchange; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D013552:Swine
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase; D018833:Chaperonins