Effect of different isolations for nucleic acid and protein complex on affinity of enriched library in SELEX experiment / 第三军医大学学报

Objective To compare the effect of different partition of nucleic acid protein complex on the affinity of enriched library in systematic evolution of ligands by exponential enrichment (SELEX). Methods Two protocols were adopted to select the enriched library to transforming growth factor ? receptor Ⅱ(TGF ? RⅡ). Protocol 1: protein was absorbed on the surface of 96 well plate; then, selection was carried out; the binding nucleotide acids were eluted from the supporter directly. Protocol 2: selection was done in solution; nucleotide acid protein complex was captured in nitrocellulose membrane; the binding nucleotide acids were obtained from membrane. Filter biding assay and gel shift assay were performed to detect the affinity of the enriched ssDNA library from different protocols. Results After 4 rounds of selection, the affinity to TGF ? RⅡ was obviously improved in the enriched library from protocol 2 compared with the initial library, while no such improvement was found in the enriched library from protocol 1. Conclusion In the SELEX experiment, the way of selection in solution, then partition of the binding nucleotide acids in filter is easier to enrich the binding fragment from initial ssDNA random library, compared with the way of fixation of target protein to solid supporter, then selection between the solid phase and liquid phase and elution of the binding nucleotide acids from supporter.

THE EXPRESSION OF HUMAN MD-2/GST FUSION PROTEIN IN E. coli / 解放军医学杂志

To design and construct an expression vector,PGEX-4T-1/MD-2, and to express human myeloid differentiation protein-2(hMD-2) and glutathione-S-transferase (GST) fusion protein in E. coli, the EcoRI/SalI sites and stop code were incorporated into the hMD-2 encoding fragment by PCR. After digesting with EcoRI/SalI, the hMD-2 encoding fragment was cloned into the expression vector PGEX-4T-1 at the corresponding sites. The positive clones selected with PCR and restriction endonuclease digestion were sequenced and the expression of GST/hMD-2 fusion protein in E. coli BL21 was analyzed with SDS-PAGE after induced by 0.4mmol/L IPTG for 3 to 5 hours. The hMD-2 encoding fragment containing stop code was correctly inserted into the expression vector PGEX-4T-1 and this was confirmed by PCR, double-enzyme identification and sequencing. The SDS-PAGE analysis indicated that the GST/hMD-2 fusion protein was successfully expressed in E. coli and the yield of the fusion protein was 30 percent of bacterial total protein. The construction of the expression vector PGEX-4T-1/hMD-2 and the expression of fusion protein GST/hMD-2 in E. coli would be useful for further investigation of MD-2.