Biomedical subjects

Zhimin He

Publications and source records attributed to Zhimin He.

10 recordsLinked to original sources

PubMed2006

High-yield expression in Escherichia coli of soluble human MT2A with native functions.

Metallothioneins (MTs) are a family of low molecular weight, cysteine rich heavy metal binding proteins with multifunction, such as metal detoxification and antioxidation, and are involved in a number of cellular processes including gene expression, apoptosis, proliferation and differentiation. However, high yield expression of human MT in Escherichia coli has not been established effectively. To produce large amounts of human MT protein at low cost, recombinant human metallothionein 2A (MT2A) protein with an N-terminal GST tag was successfully expressed at high levels in soluble form in E. coli and high purification of it was established by affinity chromatography under native conditions. The final yield was about 5mg of the recombinant MT2A per liter of bacterial culture with the purity of 97.9%. Chemical and functional characteristics analysis of the recombinant human MT2A exhibited intact metal binding ability, hydroxyl radical scavenging ability and significant protective role against DNA damage caused by UVC radiation. Establishment of highly purified recombinant human MT2A protein with native characteristics at low cost would improve its function study and wide applications in protecting against oxidative damage and UV radiation.

Cells, Cultured↗

PubMed2006

Multi-stage collisionally-activated decomposition in an ion trap for identification of sequences, structures and bn --> bn-1 fragmentation pathways of protonated cyclic peptides.

Cyclic penta-, hexa- and heptapeptides have been designed, synthesized and their fragmentations induced by multistage tandem mass spectrometry have been studied. Under low-energy collisionally activated decomposition (CAD), the protonated cyclic peptides mainly dissociate via ring opening pathways and the corresponding bn --> bn-1 pathways to form several sets of b ions as oxazolone rings (and b1 ions as aziridinone rings). Through repeated observation of these b ions in multistep CAD experiments, accurate sequencing and head-to-tail ring structure of cyclic peptides can be determined. The mistaken assignments of these b ions can be avoided by this sequencing method. Semiempirical molecular orbital calculations have been utilized to provide insight into the proposed dissociation mechanism. In addition, for cyclic peptides that include an Asn residue, the nitrogen of the Asn side chain is observed to be preferentially protonated, which can induce a unique ring-opening pathway with a loss of ammonia that competes with the conventional ring opening pathway.

Indicators and Reagents↗

PubMed2006

[Recombination of human HER2 gene encoding the extracellular domain and purification of fusion protein].

To purify the extracellular domain of HER2 in vitro and improve its prokaryotic expression abundance, the cDNA fragment encoding extracellular domain of HER2 was obtained by PCR and cloned into the expression vector pGEX-6P-1. After transforming it into Escherichia coli BL21, we instituted an investigation of different inducing conditions to try out the optimal condition for expressing soluble fusion protein. As for insoluble inclusion bodies, they were dissolved in 8 M Urea and refolded in refolding buffer. The soluble protein and the refolded protein were purified with Glutathione Sepharose 4B, respectively. The results showed that both the soluble and insoluble protein existed in Escherichia coli, but the majority was insoluble. It is beneficial to the expression of soluble fusion protein by induction at lower temperature (30 degrees C) and higher optical density (A600= 1.8) with the use of certain additive in medium. By purification of the supernatant of the lysate and refolded protein, the yield of the fusion protein was about 1.23 mg per liter culture. As a result, we have obtained the maximum soluble extracellular domain of HER2 protein, and thus have laid a foundation for further work on functional study and antibody preparation for HER2.

Cloning, Molecular↗

PubMed2006

[Preparation and quantitative analysis of methyl phenyl carbonate standard sample].

The preparation and quantitative analytical method for high-purity methyl phenyl carbonate (MPC) was developed. Dimethyl carbonate (DMC) and diphenyl carbonate (DPC) were firstly used as reactants to synthesize MPC catalyzed by TiO2/SiO2 because the disproportionation of MPC is reversible and the reverse reaction is thermodynamically favorable. The high-purity MPC standard sample was obtained by reduced pressure distillation and the removal of minor phenol with dilute sodium hydroxide solution. The qualitative analysis by gas chromatography-mass spectrometry (GC-MS) showed there were minor phenol and diphenyl carbonate in the MPC sample. The mass percent concentration of water in the MPC sample was 0.26%, which was determined by Karl Fischer titration. A gas chromatographic method with an OV-101 capillary column was established for quantifying the minor phenol and DPC in the MPC sample. The quantitative results showed the mass concentrations of phenol and DPC were 2.04% and 1.59%, respectively. The difficulty to analyze MPC, the intermediate product of the transesterification of DMC and phenol, was solved with the self-made MPC standard sample.

English Abstract↗

PubMed2005

A colorimetric method for point mutation detection using high-fidelity DNA ligase.

The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.

Cell Line↗

PubMed2005

Two new plate nozzles for the production of alginate microspheres.

Combining the Rayleigh-type jet break-up and two new plate nozzles, the alginate microsphere was produced. Spray generators made of syringe needle and laser-drilling nozzle plate and synthetic red stone nozzle plate were fabricated and contrasted. The above two plate nozzles provided lower liquid resistance and yield well. Furthermore, the more uniform microsphere was produced within a wider range of frequency by plate nozzles. Experiments using multiple-nozzle synthetic red stone plate was easy to feasible.

Alginates↗

PubMed2005

[Quantitative analysis of complicated casein hydrolysates by high performance size exclusion chromatography].

The enzymatic hydrolysates of casein are so complicated that there has no effective method for quantitative analysis. On the basis of membrane separation and high performance size exclusion chromatography (HPSEC), standard peptides with different relative molecular mass ranges were prepared, and the linear relationships between mass concentration of the standard peptides and the corresponding peak areas were established. Consequently, mass concentration of the different hydrolysates at different reaction time can be accurately calculated. The combination of chromatography and membrane separation is of great importance to the quantitative analysis of the complicated hydrolysates, which can be also applied to the other macromolecular systems, such as carbohydrates.

Amino Acid Sequence↗

PubMed2004

[Cloning, expression and purification of rabbit metallothionein-I gene in Escherichia coli].

The cDNA encoding the rabbit metallothionein-I was amplified by RT-PCR from the rabbit liver induced by cadmium and cloned into prokaryotic fusion expression vector pQE40. Then it was transformed into Escherichia coli M15. Positive expression clones were detected by colony blotting. Target protein solubility was determined by Western blotting analysis. The optimal induction condition of the level of protein expression with IPTG induction was established by SDS-PAGE electrophoresis and ImageMaster VDS software analysis. The fusion protein can be purified from lysates with Ni-NTA agarose. We found that the fusion protein with apparent molecular weight 32 KD existed in two ways: soluble and insoluble in Escherichia coli. After 1 mM IPTG induction, the level of expression of the fusion protein increased with the prolongation of induction time and reached a peak in 9 h by ImageMaster VDS software analysis, accounting for 57.4% of all the insoluble protein. The purified fusion protein was obtained by Ni-NTA affinity chromatography. This fusion protein can be used in further studies on the preparation of MT-I protein and development of protein product.

Animals↗

PubMed2002

Alginate-konjac glucomannan-chitosan beads as controlled release matrix.

Controlled release beads were prepared by using alginate (ALG), konjac glucomannan (KGM) and chitosan (CHI). Bovine serum albumin and insulin were used as model proteins for in vitro assessments. It was observed that KGM could be contained within beads, and faintness hydrogen binding and electrostatic interaction exist between ALG and KGM by infrared spectra. Clear dents were found on the surface of beads using KGM by scanning electron microscopy. Use of KGM could help increase the payload of drug. After beads were treated by 0.1 N HCl for 4 h and put into pH 7.4 buffers, protein was released from ALG-CHI beads within 1 h, while it was lost from ALG-KGM-CHI beads for 3 h. However, the leaking of protein from ALG-KGM-CHI beads was also increased in 0.1 N HCl solution. Concentration of gelling ion had great effect on release rate and gel structure. Studies of water of hydration had shown that swelling of ALG-KGM-CHI beads was higher than that of ALG-CHI beads in acidic solution, but the opposite result was obtained in alkali solution. The result indicated that the diffusion of protein was related to the viscosity and swelling properties of KGM.

Administration, Oral↗

PubMed2002

Effect of N,N'-dinitrosopiperazine on in vitro expression of human cytochrome P450 2E1.

OBJECTIVE: To establish an in vitro heterogeneous expression model of human CYP2E1 (hCYP2E1) cDNA and investigate the effect of the chemical carcinogenic N, N'-dinitrosopiperazine (DNP) on the expression of CYP2E1. METHODS: Exogenous hCYP2E1 was introduced into the mouse derived NIH3T3 cells using the lipofectamine transfection technique. Integration of exogenous hCYP2E1 gene was identified by PCR and Southern blot. After treatment with various concentration of ethanol and DNP on the transfected NIH3T3 cell cultures, RT-PCR and Western blot was applied to detect the expression level of CYP2E1. RESULTS: Two cell clones with integration and stable expression of exogenous hCYP2E1 were obtained and designated as NIH3T3-2E1-A4 and NIH3T3-2E1-A8 respectively. The expression of both hCYP2E1 mRNA and protein products was promoted after either ethanol or DNP treatment. CONCLUSION: The results suggested that the promoted expression of hCYP2E1 induced by DNP and /or ethanol is due to enhanced transcription. The mechanism of DNP carcinogenes is might be related to this in situ activated metabolism by CYP2E1.

3T3 Cells↗