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