Pig kidney dopa decarboxylase. Structure and function

Pig kidney dopa decarboxylase. Structure and functio

Protein minimization: characterization of the synthetic cyclic dodecapeptide corresponding to the reactive site region of the oil rape trypsin inhibitor type-III

The design of minimal units required for enzyme inhibition is a major field of interest in structural biology and biotechnology. The successful design of the cyclic dodecapeptide corresponding to the Phe17–Val28 reactive site amino acid sequence of the lowmolecular-mass trypsin inhibitor RTI-III from Brassica napus (micro-RTI-III) and of the recombinant murine dihydrofolate reductase-(DHFR-)micro-RTI-III fusion protein (DHFR-micro-RTI-III) is reported here. Micro-RTI-III was synthesized using a stepwise solid-phase approach based on the standard Fmoc chemistry, purified by RP-HPLC, and oxidatively refolded. DHFRmicro- RTI-III was expressed in Escherichia coli, purified bymetal-chelate affinity chromatography , and oxidatively refolded. The affinityof micro-RTI-III for bovine trypsin (Kd ¼ 1:6 109 M) is similar to that determined for DHFR-micro-RTI-III (Kd ¼ 6:3 1010 M) and native RTI-III (Kd ¼ 2:9 1010 M), at pH 8.2 and 22.0 C. Remarkably, micro-RTI-III protects the DHFR domain of DHFR-micro-RTI-III from trypsin digestion. Micro-RTI-III is a new minimal trypsin inhibitor and may be regarded as a tool in protein structure–function studies and for developing multifunctional and multidomain proteinase inhibitors

Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study

A previous case-control study reported that an in-vitro interferon (IFN)-gamma response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-gamma ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91% and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides