Abstract
The E1 alpha and E1 beta subunits of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus were produced from two genes overexpressed separately in Escherichia coli. A functional E1 enzyme was generated from disrupted mixtures of cells containing the separately overexpressed E1 alpha and E1 beta genes. The purified E1 enzyme exhibited an apparent molecular mass of 150,000 Da, consistent with an alpha 2 beta 2 structure. The Km for pyruvate and kcat (30 degrees C) were found to be 0.9 +/- 0.2 microM and 0.47 +/- 0.03 s-1, respectively. The purified E1 alpha subunit existed as a monomer (42,000 Da), whereas the E1 beta subunit existed mainly (95%) in a tetrameric form (145,000 Da). Mixing equimolar amounts of the pure recombinant E1 alpha and E1 beta subunits in vitro generated a functional E1 enzyme with a molecular mass and an E1 activity similar to those of the E1(alpha 2 beta 2) enzyme purified from disrupted mixtures of cells containing individually expressed subunits. Mixing individual subunits in vitro with one of the subunits in excess resulted in complete assembly of the lesser subunit into the intact E1 (alpha 2 beta 2) enzyme. Thus, no chaperonin is needed in vitro to promote the assembly of the separate subunits to form the E1 component of the pyruvate dehydrogenase multienzyme complex of B. stearothermophilus.
Highlights
Subunits of the Pyruvate Dehydrogenase Complexof Bacillus stearothermophilus and Assemblyof a FunctionalE l Component
How- followed by a PorosTMQ/Mcolumn (Fig. 3).Excess E1P subunit ever, all effortsto generate the appropriatpelasmid did not coelute with the intactE l enzyme, but emerged later in were unsuccessful, suggesting that something intrinsic to the a position similar to thatof the E1P subunit when it was pugenes or the El(a,P,) protein is toxic to the E. coli cells (data rified alone (Fig. 3, lane 2 )
The construction of the expression vectors pKBstEla and Thepure recombinant E l enzyme was passed through a pKBstElb for the matureEla and E1P subunits, respectively, calibrated SuperdexTM200HR gel filtration column
Summary
Annealed to the region corresponding to bases 2543-2566 and 26892705, respectively, of the DNA insert in the pBst vector. Purification of Recombinant El Enzyme a n d Its Separate Subunits, from 5% polyacrylamide gel electrophoresis(Sambrooketal.,1989). Fractions (5 ml) containing the recombinant El enzyme were pooled, dialyzed against buffer A, and loaded in batches of 3 4 mg of total protein a t 25 "C onto a Porosm Q/Manion-exchange column (0.46 x 10 cm; PerSeptive Biosystem Inc.) equilibrated withbuffer A. Fractions (0.25 ml) containing therecombinant E l enzyme were pooled; dialyzed against 100 m potassium phosphate, pH7.0 (buffer B); and stored frozeant -80 "C. For the recombinant E1P subunit, after the DE52 column chromatography, fractions (5 ml) containing this subunit werepooled, dialyzed overnight against buffeCr, and loaded in batcheofs 3-4 mg of. The recombinant E1P chain eluted with a linear gradienoft 0.3-0.5 M NaCl i n buffer A at 1 mumin over 6 ml.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
