WD40 proteins belong to a big protein family with members identified in every eukaryotic proteome. However, WD40 proteins were only reported in a few prokaryotic proteomes. Using WDSP (http://wu.scbb.pkusz.edu.cn/wdsp/), a prediction tool, we identified thousands of prokaryotic WD40 proteins, among which few proteins have been biochemically characterized. As shown in our previous bioinformatics study, a large proportion of prokaryotic WD40 proteins have higher intramolecular sequence identity among repeats and more hydrogen networks, which may indicate better stability than eukaryotic WD40s. Here we report our biophysical and structural study on the WD40 domain of PkwA from Thermomonospora curvata (referred as tPkwA-C). We demonstrated that the stability of thermophilic tPkwA-C correlated to ionic strength and tPkwA-C exhibited fully reversible unfolding under different denaturing conditions. Therefore, the folding kinetics was also studied through stopped-flow circular dichroism spectra. The crystal structure of tPkwA-C was further resolved and shed light on the key factors that stabilize its beta-propeller structure. Like other WD40 proteins, DHSW tetrad has a significant impact on the stability of tPkwA-C. Considering its unique features, we proposed that tPkwA-C should be a great structural template for protein engineering to study key residues involved in protein-protein interaction of a WD40 protein.
BjMT2 cDNA clone gene isolated from Brassica juncea was constructed in pETM-20 and expressed in E.coli as a TrxA::BjMT2 fusion protein. After affinity chromatography and cleavage from the TrxA domain, pure BjMT2 protein was obtained which strongly reacted with the thiol reagent monobromobimane. The amino acid sequence determined by mass spectrograph revealed the polypeptide contains 80 amino acids and is full of 8 and 6 cysteines with CC, CXC and C-XX-C motifs clustered near -N and -C terminus, respectively.
The expression and functional analysis of plant metallothionein type 2 from Brassica juncea were achieved in E. coli. The BjMT-2 cDNA was cloned into the pETM-20 vector fused to the Thioredoxin gene and induced to E. coli. Northern blot and SDS PAGE analysis showed, respectively, that the transcription of the TrxA::BjMT-2 fusion gene and the expression of the fusion protein were predominately in E. coli cells after IPTG induction. The BjMT-2 in the crude extraction prepared from the E. coli cells bearing TrxA::BjMT-2 vector were reacted with the antibody directed against BjMT-2. The E. coli cells exhibited an increased tolerance against Cu2+ and Cd2+ after BjMT-2 expression.
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