Media and cultures.
All strains were routinely grown using Luria-Bertani (LB) liquid broth or agar (27
) at 20°C, 30°C, or 37°C. All media were supplemented with appropriate antibiotics as follows: ampicillin at 100 μg/ml and chloramphenicol 4 μg/ml for maintaining the mini F′ plasmid pFOS1-lacIq or 10 μg/ml for maintaining pMAK705 constructs. Tests with BL21(DE3) glmS-CBD
were performed on tryptone broth or agar (LT) containing 10 g tryptone, 5 g NaCl, and 15 g agar (22
), supplemented with appropriate antibiotics and 200 μg/liter N
-acetylglucosamine, when necessary, and incubated at 30°C. The AceE-CBD in vivo
activity analysis was carried out using M9 minimal medium agar supplemented with 0.2% Casamino Acids, 1 mM MgSO4
, 0.3 mg/ml thiamine hydrochloride, 20 μg/ml tryptophan, 0.2% glucose, and, when indicated, 2 mM potassium acetate. The medium used in fermentations was as follows (per liter): 20 g animal-free soy peptone, 10 g yeast extract, 10 g NaCl, 30 g glycerol, 0.75 g KH2
, 0.75 g Na2
, 0.70 g NH4
Cl, 0.085 g K2
, 0.62 g MgSO4
O, 0.0125 g Fe(III) citrate, 0.015 g MnCl2
O, 0.0013 g Zn(CH3
O, 0.0025 g H3
, 0.0025 g Na2
O, 0.0025 g CoCl2
O, 0.0015 g CuCl2
O, 0.0014 g Na2
EDTA, and 0.05 g PPG Lumulese antifoam.
The fermentation conditions [for the expression of GluRS in BL21(DE3), NiCo21(DE3), and NiCo22(DE3) strains carrying pET21a-gluRS] were as follows. Ten-liter batch fermentations were performed using Bioflo 3000 fermentors from New Brunswick Scientific. The pH was controlled at 7.00 with automatic addition of 28% NH4OH and 46 N H3PO4. The dissolved oxygen was kept above 20% of air saturation using proportional, integral, and differential control (PID) control of agitation (400 rpm to 600 rpm) and mixing pure oxygen with air (the gas flow rate was 0.5 vessel volume per minute [vvm]). The temperature was controlled at 30°C for both growth and induction. Once the culture reached an optical density at 600 nm (OD600) of 8 to 10, isopropyl-β-d-thiogalactopyranoside (IPTG) was added to give a final concentration of 20 μM and the culture was incubated for an additional 3 h.
For shake flask expression of AlaRS in BL21(DE3) slyD-CBD, cells carrying pQE30-alaRS and pFOS1-lacIq (which provides a source of Lac repressor to control expression from the T5-lacO promoter) were grown in LB medium supplemented with 100 μg/ml of ampicillin. After outgrowth at 37°C to an OD600 of ≈0.8, 500 μM IPTG was added and the cultures were incubated for 20 h at 20°C (OD600, ≈3.2 to 3.6; cell pellet, ≈5.5 to 6 g from 1 liter culture).
For shake flask expression of AlaRS in BL21(DE3), NiCo21(DE3), and NiCo22(DE3), cells were grown in 500 ml of LB medium supplemented with 100 μg/ml of ampicillin. After incubation at 37°C to an OD600 of ≈0.8, 200 μM IPTG was added and the cultures were incubated for 4 h at 30°C (final OD600, ≈2.2 to 2.6; cell pellet, ≈2 g).
For shake flask expression of GluRS in BL21(DE3), NiCo21(DE3), and NiCo22(DE3), cells carrying pET21a-gluRS were grown in 500 ml of LB medium supplemented with 100 μg/ml of ampicillin. After growth at 37°C to an OD600 of ≈0.5, 20 μM IPTG was added and the cultures were incubated for 4 h at 30°C (final OD600, ≈2.4 to 2.6). Cells containing pET21a were grown using the same procedure in order to prepare mock lysates for the lysate-mixing experiment (see ).
Fig. 7. Utility of the NiCo strains for poorly expressed proteins. Coomassie blue-stained SDS-PAGE gel of samples taken during the purification of GluRS on Ni-NTA superflow resin (2 ml) followed by 30 min of batch incubation on chitin beads (2 ml). The purifications (more ...) Enzymes and reagents.
Restriction enzymes and DNA-modifying enzymes were provided by New England BioLabs. Mutagenesis was carried out using a Phusion site-directed mutagenesis kit (New England BioLabs). DNA amplification procedures utilized either Phusion or Taq DNA polymerases.
Strains and plasmids.
Bacterial strains and genotypes are listed in . Oligonucleotides and plasmids are described in Tables S1 and S2, respectively, in the supplemental material. The pMAK-CBD
vector for 3′ end gene tagging was created by inserting the CBD
open reading frame (ORF) from vector pTYB1 (New England BioLabs) into the polylinker region of pMAK705 (14
). An amino acid linker region which encodes LQASSS(N)10
LQS, where the first LQ codons correspond to a PstI restriction site and the last LQS codons contain a SalI restriction site, was inserted. A unique AsiSI restriction site was inserted after the CBD
ORF. All allele exchange procedures utilized derivatives of pMAK-CBD
containing DNA fragments amplified from the BL21(DE3) chromosome. Genes of interest were cloned into HindIII, SphI, PstI, and/or SacI sites. Downstream DNA (3′ flanking sequence) was cloned into AsiSI, Acc65I, SacI, and/or EagI sites.
To create the strain BL21(DE3) slyD-CBD
was transformed into BL21(DE3) and individual clones were grown in LB liquid medium with 4 μg/ml of chloramphenicol (Cm). The allele exchange method of Hamilton et al. (14
) was followed to replace the wild-type (wt) slyD
gene with the slyD-CBD
allele. The strains positive for allele exchange were cured of the pMAK vector carrying the wild-type slyD
gene using a coumermycin treatment (8
). PCR amplification with primers 4059For and 4060Rev confirmed that the slyD-CBD
allele was present at the correct locus within the chromosome of BL21(DE3) slyD-CBD
To create NiCo21(DE3), the allele exchange procedure was carried out in the same manner as that described for slyD-CBD except (i) can locus analysis was accomplished using primers 4841For and 2187Rev, (ii) analysis of the arnA-arnD locus to confirm the arnA-CBD-arnD allele was accomplished using primers 5032For and 5031Rev, and (iii) analyses to confirm replacement of the wild type glmS gene by glmS6Ala (glms allele with six histidine-to-alanine substitutions) were accomplished using primers glmSdown-Rev, glmS466check-Rev, Hind-glmS-For, and glmS-Sac-Rev.
To create NiCo22(DE3) from NiCo21(DE3), the pMAKaceE-CBD allele exchange construct was utilized. Primers 4845For and 0076Rev were used to PCR amplify the aceE-CBD-aceF locus for sequence characterization.
Mutant glmS constructs were created from pMAKglmS using a Phusion site-directed-mutagenesis kit (New England BioLabs). The glmS2Ala gene has four mutations resulting in alanine codons at positions 62 and 65 (GCTCCTCTGGCT, modified from the wt sequence CATCCTCTGCAT). The pMAKglmS6Ala construct was generated in 3 steps. First, the plasmid pMAKglmS2Ala was amplified with primers His432Ala-Rev and His436Ala-For, followed by ligation to circularize the linear PCR product. The resulting glmS4Ala mutant has the mutated sequence GCTGACATTGTGGC, which results in additional alanine codons at positions 432 and 436. Second, the pMAKglmS4Ala mutant template was amplified with primers His466Ala-Rev and glmS467-For, followed by the ligation of the linear PCR product, to generate the plasmid pMAKglmS(His62-65-432-436-466Ala). Third, the pMAKglmS(His62-65-432-436-466Ala) template was amplified with the primers glmS466-Rev and His467Ala-For, followed by ligation of the linear PCR product, to generate the plasmid pMAKglmS6Ala. The glmS6Ala allele has additional alanine codons at positions 466 and 467 (GCTGCCGCG).
was generated by PCR amplification of the gene E
x174 genomic DNA (laboratory stock) with the primers Hind-pE
-For and pE
-Xba-Rev and then cloned into the HindIII and XbaI sites of pMS119 (11
All the plasmids were confirmed by DNA sequencing using gene-specific primer s1260 (Ptac forward), s1233 (pMAK705 polylinker forward), or s1224 (pMAK705 polylinker reverse).
Ni2+ affinity chromatography using an AKTA fast-performance liquid chromatography (FPLC) system.
For the purification of AlaRS from BL21(DE3) slyD-CBD, the cell pellet was resuspended in 30 ml of buffer A1 (50 mM HEPES, pH 7.6, 1 M NH4Cl, 10 mM MgCl2, 7 mM β-mercaptoethanol, 5% glycerol, and 25 mM imidazole) supplemented with 3 ml BugBuster (10×; Novagen) and sonicated. The clarified lysate was loaded onto a 5-ml HisTrap HP column (GE Healthcare Bio-Sciences), equilibrated with 5 column volumes (CVs) of buffer A1. The flowthrough was collected, and the column was washed with 15 CVs of buffer A1. Elution was completed using a gradient from 25 mM to 250 mM imidazole in buffer B1 (50 mM HEPES, pH 7.6, 0.1 M KCl, 10 mM MgCl2, 7 mM β-mercaptoethanol, and 250 mM imidazole). Eleven CVs were collected in 28 tubes of 2 ml each.
For the Ni-NTA affinity test with GlmS, GlmS2His-Ala, and GlmS6His-Ala, cell pellets were resuspended in 6 ml of buffer A2 (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 20 mM imidazole) supplemented with 600 μl BugBuster (10×; Novagen) and sonicated. Clarified lysates were loaded onto a 1-ml HisTrap HP column, equilibrated with 5 CVs of buffer A2. After collecting the flowthrough, the column was washed with 15 CVs of buffer A2 and then eluted using a gradient from 20 mM to 400 mM imidazole with buffer B2 (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 400 mM imidazole). A pool of eluted fractions was concentrated using a Vivaspin column (Vivascience) and analyzed on SDS-PAGE gel stained with Coomassie blue R-250.
For the purification of AlaRS from BL21(DE3), NiCo21(DE3), and NiCo22(DE3) strains, the cell pellets were resuspended in 20 ml of buffer A2 (20 mM sodium phosphate, pH 7.4, 500 mM NaCl, and 20 mM imidazole) supplemented with 2 ml BugBuster (10×; Novagen) and sonicated. Clarified lysates were loaded onto a 5-ml HisTrap HP column equilibrated with 5 CVs of buffer A2. The flowthrough was collected, and the column was washed with 15 CVs of buffer A2. Elution was completed using a gradient from 20 mM to 400 mM imidazole (10 CVs collected in 25 tubes of 2 ml each) with buffer B2.
Ni2+ affinity chromatography-batch process.
For the purification of GluRS from BL21(DE3), NiCo21(DE3), and NiCo22(DE3) strains, a cell pellet of 1 g was resuspended in 10 ml of buffer A3 (50 mM sodium phosphate, pH 8, 300 mM NaCl, and 10 mM imidazole) supplemented with 1 ml BugBuster (10×; Novagen) and sonicated. The clarified lysates were loaded onto a 1-ml Ni-NTA column (Qiagen superflow resin), previously equilibrated with 10 CVs of buffer A3. After batch incubation for 1 h at 4°C with gentle shaking, the flowthrough was collected after a short spin and the resin was washed with 10 CVs of buffer A3. Elution was completed with 5 ml buffer B3 (50 mM sodium phosphate, pH 8, 300 mM NaCl, and 250 mM imidazole).
Chitin affinity chromatography.
The elution fractions obtained after Ni2+ affinity chromatography were pooled, when necessary, and incubated with 1, 2, or 5 ml of chitin beads (New England BioLabs), previously equilibrated with 5 CVs of buffer C (50 mM sodium phosphate, pH 7.4, 500 mM NaCl) at room temperature. After gentle shaking for 30 min at 4°C, the target protein was eluted from the chitin bead slurry by gravity flow. The chitin beads were then washed with 5 CVs of buffer C, and a chitin bead sample was removed for analysis of bound contaminating proteins.
SDS-PAGE and Western blot analysis.
Whole cells or purified protein fractions were prepared for SDS-PAGE analysis by mixing 2 parts sample with 1 part 3× sample buffer (New England BioLabs). Samples were analyzed by 4 to 20% SDS-PAGE. Proteins were visualized by Coomassie blue R-250 staining, or proteins were transferred to nitrocellulose membrane (Millipore) for immunoblotting. CBD-tagged proteins were detected by anti-CBD monoclonal antibody (New England BioLabs), and His-tagged proteins were detected by anti-His monoclonal antibody (EMD Biosciences). Horseradish peroxidase (HRP)-linked secondary antibody, and enhanced chemiluminescence reagents were supplied by Cell Signaling Technology.
Protein samples of approximately 1 mg were added to 20 μl of trypsin reaction buffer (50 mM Tris-HCl, 20 mM CaCl2
, pH 8) and digested overnight at 37°C with trypsin (New England BioLabs) at a protein-to-protease ratio of 20:1. Online liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) analyses of digested fractions using an Agilent 6330 Ion Trap mass spectrometer with an integrated C18
chromatin immunoprecipitation-nanoelectrospray ionization (C18
ChIP/nano-ESI) interface were performed as described previously (40
). Protein separation, digestion, and peptide analysis were repeated in triplicate for each sample. The MS-MS data were analyzed by the Spectrum Mill (Rev A.03.03.084 SR4; Agilent Technologies) search engine using parameters described previously (40
), with minor modifications. Data were searched against an E. coli
BL21(DE3) database that was supplemented with the three mutant GlmS protein sequences (GlmS2Ala
, and GlmS6Ala
). The search criteria were set to allow two missed cleavages by a tryptic digest with no other protein modifications. Peptides were validated by using a reverse database search and needed to have a score 2.0 or higher than any reverse score to be valid. Proteins built from these validated peptides scoring 20 or better were considered valid identifications for Spectrum Mill. Proteins identified from one detected peptide using a single spectrum were excluded. The false-positive rate for all Spectrum Mill analyses was less than 1%.
Protein purity analysis.
Target protein purity was analyzed using the Caliper LabChip GXII protein assay. Five microliters of protein solution (1:15 lysate mixture after Ni-NTA or chitin columns) was denatured and processed according to the protocol supplied by Caliper LifeSciences. Protein signals between 14 and 200 kDa were analyzed using HT Protein Express Chip version 2 in combination with a Protein Express reagent kit.