• IMMUNE NETWORK
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• v.10 no.2
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• pp.35-45
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• 2010

Background: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. Methods: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize $V_H$ and $V_L$ fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. Results: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of $V_H$ or $V_L$ domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of VH of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. Conclusion: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

• Journal of the Korean Chemical Society
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• v.43 no.4
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• pp.461-468
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• 1999

Acetolactate synthase (ALS) catalyzes the first common reaction in the biosynthesis of branched-chain amino acids, valine, leucine, and isoleucine. ALS is the common target of several classes of structurally diverse herbicides, the triazolopyrimidines, the imidazolinones, the sulfonylureas, and pyrimidyl-oxy-benzoates. We examined ihibitory activities of newly synthesized triazolopyrimidine sulfonamide derivatives using partially purified ALS from barley. $IC_{50}$ values for the active derivatives are 0.5nM∼8$\mu$M. Among them TP1 and TP2 are the most potent ALS inhibitors with $IC_{50}$ values of 0.5nM and 1.6nM, respectively. These inhibitors are more potent in the inhibition of barley ALS than commercial herbicides, Metosulam ($IC_{50}$;3.6 nM), Flumetsulam ($IC_{50}$;126 nM), and Cadre ($IC_{50};4 {\mu}M$). The progress curves for inhibition of ALS by TP2 showed that the amount of inhibition increases with time. The inhibition of ALS by TP2 was mixed-type inhibition with respect to pyruvate. Dual inhibition analyses of TP2 versus an imidazolinone, Cadre, and Leu showed parallel and intercepting kinetic pattern, respectively. The results suggest that TP2 binds to ALS competively with Cadre but not with Leu. Chemical modification of cysteinly residues in ALS decreased the sensitivity of ALS to Leu, while the modification did not affect the sensitivity of ALS to TP2 and Cadre.

• Journal of the Korean Chemical Society
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• v.42 no.3
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• pp.306-314
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• 1998

Acetolactate synthase (ALS) is the common enzyme in the biosynthesis of branched chain amino acids, Val, Leu, and Ile in bacteria, yeast, and higher plants. The enzyme is target site of several classes of structually diverse herbicides, including the sulfonylureas, the imidazolinones, the triazolopyrimidines, and the primidyl-oxy-benzoates. We have synthesized new triazolopyrimidine (TP) derivatives, and determined their inhibitory activities on barley ALS. $lC_{50}$ values for the active compounds were 3.2 nM-0.62 mM, and some of them appeared to be potent inhibitors. The progress curves for inhibition of ALS by TP4, a representative derivative, indicated that the extent of inhibition increased with incubation time. The inhibition of ALS by TP4 showed mixed-type inhibition with respect to pyruvate. Dual inhibition analyses of TP4 versus imidazolinone Cadre and feedback inhibitor Leu suggested that three different classes of inhibitors bind to ALS in a mutually exclusive manner. Chemical modification of tyrosyl residues of ALS decreased sensitivity of ALS to TP4, while modification of tryptophan and cysteine did not affect the sensitivity.