Jianshu Chen

Selecting DNA aptamers for endotoxin separation

ObjectivesTo select aptamers for endotoxin separation from a 75-nucleotide single-stranded DNA random library using systematic evolution of ligands by exponential enrichment.ResultsAfter 15 rounds of selection, the final pool of aptamers was specific to endotoxin. Structural analysis of aptamers that appeared more than once suggested that one aptamer can form a G-quartet structure. Tests for binding affinity and specificity showed that this aptamer exhibited a high affinity for endotoxin. Using this aptamer, aptamer-magnetic beads were designed to separate endotoxin.ConclusionsUsing these aptamer-magnetic beads, a new method to separate endotoxin was developed to enable specific separation of endotoxin that can be applied to drug and food products.

IgG PURIFICATION USING AFFINITY FILTRATION WITH SULFAMETHAZINE-AFFINITY CARRIERS

Immunoglobulin G (IgG) antibodies are used extensively for analytical, diagnostic, and therapeutic applications. However, there are some disadvantages to purify IgG antibodies by protein A and G affinity chromatography. Therefore, it is necessary to find an effective alternative and nonchromatographic method to purify IgG. Dextran microparticles were activated and coupled with sulfamethazine to form sulfamethazine-affinity carriers. Then the carriers were used to purify IgG by affinity filtration. Quantitative and qualitative determination proved that sulfamethazine would successfully bond to the surface of dextran microparticles with a density of 85.5 μmol/g (wet). Affinity carriers were proved to withstand high shear force and reveal rare sulfamethazine leakage under filtration conditions between pH 3 to 11. The maximum IgG-binding capacity of affinity carriers was 8.03 mg IgG/g (wet). The affinity filtration process obtained a recovery yield above 80% and purity above 90%. Thus, this work involved in both the advantages of membrane filtration and affinity purification. The results, for the first time, proved that it is possible to use the small ligand sulfamethazine for affinity filtration of IgG. It is an attractive alternative to conventional protein A or G affinity chromatography.

Preparation of recombinant human thymic stromal lymphopoietin protein

ABSTRACT Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0 M in 50 mM Tris (pH 8.0), 1 mM EDTA, 50 mM NaCl, 10% glycerol, 400 mM L-Arg, 0.2 mM oxidized glutathione, and 2 mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52 × 10−3 mg/L.

Construction and application of an electrochemical biosensor based on an endotoxin aptamer

An electrochemical biosensor that used an aptamer as a biological element was constructed to detect endotoxin. Biolayer interferometry was used to obtain the affinity constant of an aptamer for lipopolysaccharide, which had an equilibrium dissociation constant of 22.9 nM. The amine‐terminated aptamer was then assembled on a gold electrode surface using 3‐mercaptopropionic acid as an intermediate linker. The modification of the gold electrode was confirmed by cyclic voltammetry and electrochemical impedance spectroscopy. In the range of 0.001–1 EU/mL, the increase in electron transfer resistance of the biosensor was linear with the logarithmic value of the endotoxin concentration. The constructed biosensor exhibits sensitivity and a low limit of detection.