Biomedical subjects

Li-Jun Bi

Publications and source records attributed to Li-Jun Bi.

8 recordsLinked to original sources

PubMed2006

Cell-free bioassay for measurement of dioxins based on fluorescence enhancement of fluorescein isothiocyanate-labeled DNA probe.

This study aims to develop a rapid and sensitive cell-free bioassay of dioxins. It is known that dioxin ligand can bind heterodimeric aryl hydrocarbon receptor (AhR) and triggers the formation of the complex of dioxin-AhR, AhR nuclear translocator (ARNT), and dioxin-responsive element (DRE) region of the DNA. The hypothesis of the proposed method is that if FITC were labeled at the DRE sequence, its fluorescence intensity would be enhanced when the complex forms because the interaction interface of the binding components (AhR, ARNT, and DRE) creates a rather hydrophobic condition that is in favor of FITC emission. Effects of modification site of FITC on the DNA probes on binding efficiency between the complex components and fluorescence emission enhancement were evaluated by surface plasmon resonance and fluorescence analysis, respectively. Results showed that the labeling site at the second base at the 5' end apart from the core region (5'-TNGCGTG-3') of DRE did not obviously interfere with the binding between the DNA probe and dioxin-AhR/ARNT hybrid but presented significant fluorescence emission enhancement. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the typical toxin in this study. The method had a linear range of 1-100 pM, with detection limit of 0.1 pM (0.64 fg/assay) and coefficient of variation of 5.6% (n = 10, 50 pM TCDD in transformed cytosol). The whole detection cycle was approximately 4 h. The method was also used to estimate the toxic equivalents (TEQ) of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD). Measurement of TEQs of the mixture of TCDD, PeCDD, and HxCDD were highly consistent with the predicted data. The average recovery using fly ash extract was approximately 93%.

Biological Assay↗

PubMed2006

The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.

Adenosine Triphosphatases↗

PubMed2006

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip.

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin-streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K (D) approximately equal to 4.01 x 10(-7). All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.

Animals↗

PubMed2006

Phage display mediated immuno-PCR.

Immuno-PCR (IPCR) is a powerful detection technology in immunological study and clinical diagnosis due to its ultrasensitivity. Here we introduce a new strategy termed phage display mediated immuno-PCR (PD-IPCR). Instead of utilization of monoclonal antibody (mAb) and chemically bond DNA that required in the conventional IPCR, a recombinant phage particle is applied as a ready reagent for IPCR experiment. The surface displayed single chain variable fragment (scFv) and phage DNA themselves can directly serve as detection antibody and PCR template, respectively. The aim of the design is to overcome shortcoming of low detection sensitivity of scFv so as to largely facilitate the real application of scFv in immunoassay. The idea has been demonstrated by applying hantaan virus nucleocapsid protein (NP) and prion protein (PrP) as detection targets in three experimental protocols (indirect, sandwich and real-time PD-IPCR assays). The detection sensitivity was increased 1000- to 10,000-folds compared with conventional enzyme-linked immunosorbent assays (ELISAs). This proof-of-concept study may serve as a new model to develop an easy to operate, low cost and ultrasensitive immunoassay method for broad applications.

Capsid Proteins↗

PubMed2006

[Fusion tags technology and their applications].

Fusion tags are originally developed to facilitate the purification of recombinant protein from crude extracts. In recent years, the discovery of different tags and the development of fusion strategy make the function of fusion tags diversified. However, there was no a cure-all fusion tag for different applications. We here give an overview of fusion tag technology and the different applications of fusion tags, including the purification, detection and oriented immobilization of recombinant protein, the visualization of bioevent in vivo, the enhancement of the yield of protein, the improvement of the solubility and stability of the expressed protein.

Recombinant Proteins↗

PubMed2005

Construction and characterization of different MutS fusion proteins as recognition elements of DNA chip for detection of DNA mutations.

Three MutS fusion systems were designed as the mutation recognition and signal elements of DNA chips for detection of DNA mutations. The expression vectors containing the encoding sequences of three recombinant proteins, Trx-His6-GFP-(Ser-Gly)6-MutS (THGLM), Trx-His6-(Ser-Gly)6-Strep tagII-(Ser-Gly)6-MutS (THLSLM) and Trx-His6-(Ser-Gly)6-MutS (THLM), were constructed by gene slicing in vitro. THGLM, THLSLM and THLM were then expressed in Escherichia coli AD494(DE3), respectively. SDS-PAGE analysis revealed that each of the expected proteins was approximately 30% of the total bacterial proteins. The recombinant proteins were purified to the purity over 90% by immobilized metal (Co2+) chelation affinity chromatography. Bioactivity assay indicated that three fusion proteins retained the mismatch-binding activity and the functions of other fusion partners. DNA chips arrayed both mismatched and unpaired DNA oligonucleotides as well as rpoB gene from Mycobacterium tuberculosis were prepared. THGLM, THLSLM and THLM that was labeled with Fluorolinktrade mark Cy3 reactive dye, were then used as both mutation recognition and labeling elements of DNA chips. The resulting DNA chips were used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides and single base mutation in rpoB gene of M. tuberculosis that is resistant to rifamycin.

Carbocyanines↗

PubMed2003

A MutS-based protein chip for detection of DNA mutations.

This paper describes a new protein chip method for detection of single-base mismatches and unpaired bases of DNA, using a genetic fusion molecular system Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching Strep-tag II and mutS gene to pET32a (+) sequentially with insertion of a linker peptide coding sequence before and behind Strep-tagII gene, respectively. THLSLM was expressed in E. coli AD494 (DE3) and purified using Ni(2+)-chelation affinity resin. THLSLM retained both mismatch recognition activity and streptavidin binding affinity. THLSLM was then immobilized on the chip matrix coated with streptavidin through the Strep-tag II-streptavidin binding reaction. The resulting protein chip was used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides, as well as a single-base mutation in rpoB gene from Mycobacterium tuberculosis, with high specificity. The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations.

Adenosine Triphosphatases↗

PubMed2002

[High expression and identification of DNA mismatch repair gene mutS in Escherichia coli].

DNA mismatch repair gene mutS (2.56 kb) was PCR modified and cloned into a secretive prokaryotic expression vector pET32a (+) which carries a N-terminal His.tag + and thioredoxin sequence. MutS protein was expressed with high level after IPTG induction using the strain E. coli AD494(DE3). SDS-PAGE revealed that the expected protein with a molecular weight of 108 kD which is about 35% of the total bacterial proteins is almost soluble. The expected protein was purified directly by immobilized metal (Ni2+) chelation affinity chromatography and the purity is over 90%. MutS protein activity verified using mismatch DNA showed that the expression product can recognize and bind to base-pair mismatch specifically.

Adenosine Triphosphatases↗