Nuclear magnetic resonance (NMR), resonance Raman, and Fourier transform infrared spectroscopy (FTIR), are among the spectroscopic methods capable of probing the structure and function of multiheme cytochromes c. All stand to benefit from the spectral simplification afforded by the isotopic labeling of specific heme carbons.Material and methods Chemical reagents
All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) with the exception of the BugBuster protein extraction reagent (Merck KGaA, Darmstadt, Germany) and the Complete protease inhibitor cocktail tablets (Roche, Basel, Switzerland).Synthesis of labeled dALA
The method reported by Bunce et al. (35) was used to synthesize 1,2-13C-labeled dALA. This compound gives rise to hemes with 13C-labeled atoms in the methyl positions and also at the β- and carboxylate carbons of the propionate groups (36).Construction of the hemA knockout in E. coli strain JM109(DE3)
The E. coli strain JM109(DE3) was purchased from Promega (Fitchburg, WI, USA). In order to generate a ΔhemA mutant in E. coli JM109(DE3), the method developed by Datsenko & Wanner to disrupt chromosomal genes was used (37). First, the hemA gene was replaced with a kanamycin resistance (kan) gene, which was then subsequently excised. In order to do this, the plasmid pKD4 was used as a template and the region that contained the kan resistance gene flanked by Flp recombination target (FRT) sites was amplified. Primers homologous to the flanking sites of the kan gene were constructed. These also carried nucleotide extensions homologous to the chromosomal regions adjacent to the hemA gene (forward: 5′-CAGACTAACCCTATCAACGT-TGGTATTATTTCCCGCAGACGT-GTAGGCTGGAGCTGCTTC-3′ and reverse: 5′-GGCGTAAATGCACCCT-GTAAAAAAAGAAAATGATGTACT-GCATATGAATATCCTCCTTAG-3′). The PCR product consisted of the kan gene flanked by 40-bp sequences homologous to the regions adjacent to the hemA gene of E. coli. Before inserting the amplified region, the E. coli cells were transformed with the plasmid pKD46 in order to obtain better transformation and recombination rates. This plasmid contains the genes of the phage λ Red protein recombination system. Kanamycin-resistant clones were selected on Luria-Bertani (LB) medium supplemented with 50 mg/L dALA and 50 mg/L kanamycin. The kan gene was subsequently eliminated using the helper plasmid pCP20, which encodes for the Flp recombinase and promotes recombination at the FRT sites. This step allows the excision of the antibiotic gene from the chromosome, since it is flanked by the FRT sites, leaving behind only a small residual scar. Successful disruption of the hemA gene was verified by PCR. This hemA knockout E. coli mutant was denominated LS542.Construction of the E. coli strains used for protein expression
The plasmid pEC86 (23), containing the ccmABCDEFGH (cytochrome c maturation) genes, was introduced via electroporation into LS542 to create LS543.
Expression vectors pET21a-stC(D2N) (38) and pKP1 (39) containing the genes encoding for the mutant STC(D2N) and native MtrA, respectively, were subsequently introduced into LS543. These strains were denominated LS543-STC(D2N) and LS544, respectively.
In order to compare expression levels between the hemA knockout E. coli mutant strain and the wild-type (wt) strain E. coli JM109(DE3), the plasmids pEC86 and pET21a-stC(D2N) were also introduced into wt. This strain was denominated ROL002.
With exception of the wild-type, all the other strains were grown in media supplemented with 50 mg/L dALA. The supplementation of the media with dALA is essential for growth of the mutant strains.Optimization of protein expression
To optimize the expression, the E. coli strains LS543-STC(D2N), LS544, and ROL002 were tested using several overexpression methods for cytochromes. In all the cases, controlled modifications of temperature, dALA concentration, IPTG concentration, and induction period were performed.
The overexpression of proteins in a rich medium with an induction step at a certain OD600nm was tested (24). This was done using LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) and also in Terrific Broth (TB) medium (12 g/L tryptone, 24 g/L Yeast extract, 4 mL glycerol, 2.31 g/L KH2PO4, 12.54 g/L K2HPO4) supplemented with 35 mg/L chloramphenicol, 100 mg/L ampicillin, and 50 mg/L dALA. Cells were grown overnight at 30°C and 180 rpm, and 1% of this culture served as inoculum for the fresh medium. Batches of fresh medium were supplemented with different concentrations of dALA, ranging from 20 to 200 mg/L, in order to determine the best dALA concentration to overexpress the protein. The culture was then allowed to grow at 180 rpm, at a defined temperature (25°, 30°, or 37°C) until an OD600nm ╛ 0.8 was reached. The protein expression was induced by addition of different concentrations of IPTG, ranging from 0 to 1 mM.