Recombinant Human Anti-Piperidinium Antibody (14D9) (CAT#: PABX-155)

Figure 1 Expression and purification of 14D9-ScFv NusA fusion constructs.

Lane A: 14D9 ScFv-NusA fusion protein expressed in the cytoplasm of BL21(DE3) TrxB–, Gor– cells after purification with IMAC. Lane B: 14D9 ScFv-NusA fusion protein expressed in the cytoplasm of BL21(DE3) cells after purification with IMAC. Lane C: NusA protein expressed in the cytoplasm of BL21(DE3) cells after purification with IMAC. Lane D: Total cell extract for the expression of 14D9 ScFv without NusA in BL21(DE3) TrxB-, Gor-cells. Lane E: ScFv purified from a hapten affinity column after filtration through a Ni2+-NTA column.

Zheng, L., Baumann, U., & Reymond, J. L. (2003). Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm. Journal of biochemistry, 133(5), 577-581.

Figure 2 Catalytic activity assay for 14D9 antibodies.

Percent conversion of enol ether 2 to ketone 3 is shown as a function of time. Measured by peak integration of RP-HPLC. Conditions: 300 µM substrate 2 in aq. PBS with 1.5% v/v acetonitrile, 1 mg/ml protein, 25C. (solid square): catalytic reaction of 14D9 ScFv-nusA fusion protein; (open square): inhibition of 14D9 ScFv-nusA fusion protein with 50 µM hapten 1b; (solid triangle): catalytic reaction of 14D9 Fab purified from hybridoma; (open triangle): inhibition of 14D9 Fab with 50 µM hapten 1b; (solid circle): catalytic reaction of 14D9 ScFv cleaved from the 14D9 ScFv-nusA fusion protein; (open circle): inhibition of 14D9 ScFv with 50 µM hapten 1b.

Zheng, L., Baumann, U., & Reymond, J. L. (2003). Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm. Journal of biochemistry, 133(5), 577-581.

Figure 3 Enantioselectivity assay for 14D9 antibodies.

The peak of product ketone 3 from the analytical RP-C18 column was collected and lyophilized, and the residue taken was up in hexane/isopropanol = 1:1, and analyzed on a chiral-phase column (Chiralpak OD, Daicel, 22 cm 0.45 cm), which allows the separation of the two enantiomeric products (eluent: hexane/isopropanol=7:3; 1 ml/min) as tR(R)-3 = 8.6 min, tR(S)-3 = 12.4 min. (A) racemic ketone 3 produced by treatment of enol ether 2 with 0.1% TFA, R/S = 49%/51%. (B): (S)-ketone 3 from the reaction with 14D9-Fab, R/S = 6%/94%; (C): (S)-ketone 3 from the reaction with 14D9 ScFv-nusA fusion protein, R/S = 8%/92%; (D): (S)-ketone 3 from the reaction with 14D9 ScFv protein, R/S = 3%/97%.

Zheng, L., Baumann, U., & Reymond, J. L. (2003). Production of a functional catalytic antibody ScFv-NusA fusion protein in bacterial cytoplasm. Journal of biochemistry, 133(5), 577-581.