ARPA-E REMOTE Q10 report

Molecular cloning and sequencing of designed hydratase enzymes

Synthetic genes coding for 12 designed hydratase proteins based on 5 naturally-occuring enzymes were synthesized as linear dsDNA by Life Technologies. Constructs were cloned into expression vector pET29b(+) using Gibson assembly. Table 1 shows the progress of 12 constructs. Sequence verification for 10 constructs is in progress, and 2 constructs are currently sequence verified.

Protein expression and purification of verified constructs

Small-scale protein expresion

For small-scale expression experiments, 2 sequence-verified constructs were transformed into Escherichia coli BLR, and single colonies were used to inoculate 25 mL of Terrific Broth in 50 mL Falcon tubes. In parallel, cells were grown in 25 mL cultures in 250 mL flasks. No difference in pellet weights or soluble protein expression as assessed by SDS-PAGE was observed (data not shown) between these two growth methods.

Large-scale protein expresion

Currently, we are testing the expression of these proteins in large-scale 500 mL cultures to see if higher yields can be obtained. We expect to have small- and large-scale expression data for each of these 12 proteins in the next quarterly report.

Designed hydratase enzymes. Progress of 12 designed hydratase constructs
Construct name Protein yield (mg/mL) Status
1bvv_A 0 Small-scale testing complete
1bvv_C Sequence verification
1fcy_3Tyr Sequence verification
1fcy_A Sequence verification
1fcy_C Sequence verification
1qv0_A Sequence verification
1qv0_C Sequence verification
1ukb_A Sequence verification
1ukb_C 0.1 Small-scale testing complete
1upv_A Sequence verification
1upv_C Sequence verification
1r5l_A Sequence verification
1r5l_C Sequence verification

Detection of 2-allylphenol by GC-FID

Assay development and estimate of limit of detection

Assay development was conducted with samples containing 1 mM 2-allylphenol (Sigma) in DMSO (Sigma) prepared fresh on the same day as GC-FID detection.

An Agilent 7820A gas chromatograph fitted with HP-5 column with the following settings was chosen: FID temp=250C, inlet temp=250C, injection volume=2µL, mode=50:1 split. A limit of detection of 1 µM 2-allylphenol in DMSO was determined by determining the FID response to 6 concentrations of 2-allylphenol: 1 mM, 100 µM, 10 µM, 1 µM, 100 nM, and 0 (DMSO only). All standard curve experiments were performed in triplicate.

Standard curve under enzyme conditions

Vials containing 500 µL of a solution of 50 mM HEPES buffer, 150 mM NaCl, 2% DMSO, and range of concentrations of 2-allylphenol (1 mM, 500 µM, 250 µM, 125 µM, 0) at pH 7.50 were extracted with 500 µL DMSO, vortexed for 1 sec, and centrifuged for 5 min at 4,700 RPM. An Agilent autosampler was used to inject 2 µL for each run.

Figure 1: standard curve of 2-allylphenol detection by GC-FID. Detection of 2-allylphenol by GC-FID was found to be linear in the range 0–1 mM, with a slope of 0.74 pA/µM and a y-intercept of 0.215 pA.

Pilot enzyme assays using 2 sequence-verified constructs

The above protocols were used to assess the enzyme activity of 2 constructs, 1bvvA and 1ukbC. Biological duplicates were produced in 25 mL cultures, and proteins purified via immobilized metal ion chromatography using cobalt resin (Thermo HisPur).

A 500 µL solution of purified enzyme that had undergone buffer exchange at ~0.05 mg/mL was spiked with 10 µL of a 1 M solution of 1 M 2-allylphenol in DMSO, resulting in a ~500 µL enzyme reaction containing ~0.05 mg/mL purified enzyme, 50 mM HEPES, 150 mM sodium chloride, trace imidazole, 1% DMSO, and 1 mM 2-allylphenol. Reactions were prepared in glass vials (Fisher) with Teflon-coated septa (Fisher) and incubated at 18 C for 24 hours with gentle rocking.