Study on metal binding capacity of the freshwater crab Sinopotamon henanense's recombinant copper specific binding metallothionein expressed in Escherichia coli.

The copper specific binding metallothionein (CuMT) is a type of cysteine-rich, metal-binding, small protein which plays an important role in Cu metabolism in vertebrates. In this study, we investigated the metal tolerance and removing ability of recombinant strains harboring CuMT obtained in vivo from the freshwater crab Sinopotamon henanense (ShCuMT) in order to study its physiological functions and metal binding capacity. We performed a 3D modeling of ShCuMT and created its structural and functional models using the I-TASSER program.

Analysis of spectrometry and thermodynamics of the metallothionein in freshwater crab Sinopotamon henanense for its binding ability with different metals.

The metal binding nature of heterologously expressed metallothionein of Sinopotamon henanense (ShMT) had been demonstrated previously. In this study, we analysed the stoichiometry of ShMT yielded in vivo and exchange reactions of the Zn-ShMT with Cd, Pb and Cuin vitro via electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), and isothermal titration calorimetry (ITC). The results of ESI-TOF-MS analyses showed that metal-ShMT synthesized in vivo had three major forms, namely Zn-, Cd-, and Pb-ShMT.

A modified hybridization protocol for low-density diagnostic DNA microarrays.

To promote the application of DNA microarrays for clinical diagnosis, the problems of cross-hybridization and low signal intensity in the hybridization processes has been addressed. We tested a new hybridization protocol for low-density diagnostic DNA microarrays, by skipping the purification step during sample labeling, while elevating the hybridization temperature from 42 degrees C to 52 degrees C, adding a step of distilled water rinsing immediately after hybridization and before the low stringency washing steps. It was found that the modified hybridization protocol works well in our study, which increased detection sensitivity and eliminated nonspecific signals.

A modified restriction display PCR method in sample-labelling of DNA microarray.

The restriction display PCR is a useful technique for studying the diversity of gene expression. This method involves ligating the digested genes with adapters and amplifying the gene fragments by PCR using universal and selective primers. In this study, we improved this restriction display PCR method by using Cy3-UP, a fluorescently labelled universal primer, in place of Cy3-dCTP in sample-labelling for DNA microarray.