Materials Restriction enzymes, β-agarase, DNA polymerases, T4 ligase, antarctic phosphatase, Litmus 38, the pMAL Protein Fusion and Purification System including pMAL-c2X, pMAL-c2G, and pMAL-c4X, the USER Friendly Cloning kit, amylose resin, anti-MBP monoclonal antibody linked to horseradish peroxidase, and synthetic oligonucleotides were obtained from New England Biolabs. The nuclease Benzonase from Serratia marcescens was purified as an MBP fusion protein and separated from MBP by digestion with factor Xa protease, using the pMAL system (data not shown). Whatman Unifilter 800 microplates with filter bottoms and Immulon 2HB microplates were purchased from VWR. The MinElute DNA Extraction and QIAquick Spin kits were purchased from Qiagen. The detergent MEGA 10 was purchased from Dojindo. Hen egg white lysozyme and Coomassie brilliant blue R were purchased from Sigma–Aldrich. Disposable polypropylene columns (#732-6008) were purchased from BioRad; 10–20% gradient gels were purchased from either Daiichi or Invitrogen/Novex.
Strains and plasmids
Host strains TB1, ER1992, ER2502, ER2984, NEB 5-alpha, NEB Turbo, and NEB Express were obtained from New England Biolabs. The source of the Bacillus circulans
chitin binding domain (CBD) was pMB50, kindly provided by M. Xu (see Supplemental data
). The construction of the pMAL derivatives pSN1578 and pIH1684 is described in the supplemental data. The pMAL-DHFR and pMAL-GAPDH plasmids were constructed using a protocol similar to ligation-independent cloning (Aslanidis and de Jong 1990
), except in this case, the overhangs created were short and still require ligase to produce a transformable plasmid (see Supplemental data
). The DHFR and GAPDH regions from the resulting plasmids were then transferred into other pMAL derivatives as AvaI, SbfI or AvaI, HindIII fragments.
Random PCR mutagenesis was carried out as described in (Fromant et al. 1995
). Briefly, four reactions were carried out, each with three of the four nucleotides at a concentration of 0.2 mM and the fourth nucleotide at 0.5 mM. PCR was performed using Taq polymerase in the presence of 6 mM MgSO4
. For the first library, the malE
gene was amplified with the primers 5′GGAGACAUGAATTCAATGAAAATCGAAGAA and 5′GGGAAAGUAAGCTTAATCCTTCCCTCGATC, using pMAL-c2X as a template. PCR fragments were cloned into linearized pNEB208A using the USER Friendly Cloning Kit, following the manufacturer’s instructions. For the second library, the malE
gene was amplified with the primers 5′CACGAGCAATTGACCAACAAGGAC and 5′GATCGAGAGCTCGAATTAGTCTGC. Both the PCR product and pIH1684 were cut with MfeI and SacI and gel purified, and the two fragments were ligated. Transformants from each library were grown overnight in 0.9 mL LB + 10 uM IPTG and 100 ug/mL ampicillin and then lysed by adding 0.1 mL of a detergent/lysozyme/nuclease solution, giving a final concentration of 50 mM Tris–Cl pH 8.0, 50 mM NaCl, 0.2 mM EDTA, 0.2 mg/mL lysozyme, 0.3% MEGA-10, and 20 U/mL of Benzonase (to reduce viscosity; modified Kunitz units (Friedhoff et al. 1994
)) and incubating for 2 min at room temperature.
Screening MBP mutants by affinity purification
Of the extracts prepared, 0.7 mL, as described above, was applied to 100 uL of amylose resin in a well of a Unifilter 800 microplate, and each well was washed with 2 mL of 20 mM Tris–Cl, 0.2 M NaCl, 1 mM EDTA, pH 7.4 (column buffer, CB) containing 0.2% Tween 20, then with 2 mL of CB without Tween 20, and finally with 1 mL of 10 mM sodium phosphate, 0.2 M NaCl, 1 mM EDTA, pH 7.2. The protein bound to the amylose resin was then eluted with 0.2 mL of 10 mM maltose, 10 mM sodium phosphate, 0.2 M NaCl, 1 mM EDTA, pH 7.2.The eluate was transferred to an Immulon 2HB microplate and incubated overnight at 4°C. The microplate wells were then emptied, washed twice with 20 mM Tris–Cl, 150 mM NaCl, pH 7.5 (TBST), then blocked with 0.36 mL TBST
3% bovine serum albumin for 1 h at 37°C. The wells were washed twice with TBST, then 0.2 mL of a 1:2,000 dilution of anti-MBP monoclonal antibody linked to horse radish peroxidase in TBST
3% bovine serum albumin was added to each well and the plate incubated at 37°C for 1 h. The wells were emptied and then washed three times with TBST. The wells were developed with 0.01% o
-phenylenediamine, 0.003% hydrogen peroxide in water. The detection reaction was stopped by adding 0.025 mL 4 M H2
, and wells were assayed spectrophotometrically at 490 nm. Cells were recovered from samples corresponding to lysates that showed higher binding and elution as compared to wild-type MBP. These candidates were grown and retested to confirm the higher binding and elution. A list of the mutations is given in Table S1
Subcloning and separation of mutations
For the first library, the genes for candidate MBP mutants were processed with PCR from pNEB208A using the primers 5′ GACTCATATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGC and 5′ ATATAAGCTTTCACCTTCCCTCGATCCCGAGGT. The PCR fragment was cut with NdeI and HindIII and ligated into the pMAL derivative pIH1684 cut with the same enzymes. The second library was constructed directly in pIH1684, so testing proceeded without needing to subclone. Of the 25 mutants obtained in the screen, ten had silent mutations in addition to one or more missense mutations; the silent mutations were ignored for this analysis. Five were identical in sequence to another mutant, presumed to be siblings, and dropped. Nine had a single missense mutation. The remaining 11 had from two to five missense mutations. The mutations in the multiple mutants were reconstructed separately by either subcloning, when appropriate sites were available, or four primer mutagenesis as described in Guan and Kumar 2005
. The PCR fragments produced were ligated into pIH1684.
Small-scale affinity purification of MBP and MBP fusions
A 200-mL culture of each variant of MBP2 to be tested was grown in LB
0.1% glucose and 100 µg/mL ampicillin to early log phase, then induced by addition of IPTG to a final concentration of 0.3 mM. The cells were harvested after 2 h and resuspended in 5 mL CB, sonicated, and clarified at 9,000×g
for 30 min. A 1.6-mL sample of the supernatant was diluted to 8 mL with CB and applied to a 1-mL amylose column. The column was washed with 16 mL CB and eluted with 4 mL of CB
10 mM maltose. The protein in the eluate was quantitated by spectrophotometry at 280 nm (A280
of 1 corresponds to 0.66 mg/mL). Values for wild-type (included in all experiments as a control) are the average of 19 experiments, and values for A312V (included as an additional control in most experiments) are the average of 16 experiments; all others are the average of 2–8 experiments and are expressed as % yield relative to wild-type in the same experiment, then normalized to the overall wild-type average.
Solubility enhancement of MBP fusions
A 10-mL culture of NEB Express containing the pMAL-DHFR or pMAL-GAPDH derivative was grown to 2
cells/mL in LB
0.1% glucose and 100 µg/mL ampicillin, induced with 0.3 mM IPTG, incubated for an additional 2 h, then cells were harvested by centrifugation at 3,000×g
in a microfuge. Each pellet was resuspended in 1 mL of CB and lysed by sonication. The lysate was centrifuged for 2 m at 14,000×g
, and the supernatant was removed and designated the soluble fraction. The pellet was resupended in 1 mL of the same buffer and designated the insoluble fraction. A sample (4 uL) of each fraction was run on SDS–PAGE for each strain, the gels were dried and scanned, and the amount of MBP fusion protein in each lane was quantitated using ImageJ (Abramoff et al. 2004
). Results are expressed as a ratio of soluble MBP fusion protein to total MBP fusion protein present in the soluble and insoluble lanes.
Determination of dissociation constants
Kd measurements were made by measuring maltose-induced fluorescence quenching (Miller et al. 1983
) using a Perkin–Elmer LS50B luminescence spectrophotometer. All measurements were carried out at room temperature (25–26°C), at a protein concentration of 15 µg/mL (0.32 µM) and an excitation wavelength of 290 nm. Emission scans were done with excitation slit width of 2.5 nm and an emission slit width of 5.0 nm. Titrations with maltose or maltotriose were carried out with excitation and emission slit widths of 5.0 nm, and an emission wavelength of 330 and/or 350 nm, depending on the mutant (see “Results
”). The data were plotted as percent quenched vs. input maltose/maltotriose concentration, and the dissociation constant determined by a curve-fit using the equilibrium binding equation in Miller et al. 1983
and the software KaleidaGraph 4 (Synergy Software).