Biomedical subjects

Zhongli Ding

Publications and source records attributed to Zhongli Ding.

3 recordsLinked to original sources

PubMed2004

Smart polymer-streptavidin conjugates.

The conjugation of stimuli-responsive, or "smart," polymers to streptavidin is described. The polymer is synthesized with a thiol-reactive end-group, which is used to end-graft the polymer to cysteine or lysine side-chains that are genetically engineered into controlled positions on the streptavidin surface. The conjugation positions are chosen on the basis of their location relative to the binding site, together with the criteria that they be solvent accessible and thus reactive. The polymer composition can be controlled to impart responsiveness to temperature, pH, and/or specific wavelengths of light. These signals are sent to the polymer, which serves as an antennae and actuator to gate biotin or biotinylated protein association with the streptavidin binding sites. The molecular switching and gating activity is directed by the reversible polymer transition between a hydrophilic, expanded coil and a more hydrophobic, collapsed state that is smaller in volume. The differences in the polymer steric properties serve to block or allow ligand access to the binding site. The control of polymer molecular weight is a particularly important design parameter for these molecular gates.

Acrylamides↗

PubMed2002

Affinity separation using an Fv antibody fragment-"smart" polymer conjugate.

Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.

Acrylic Resins↗

PubMedDate not supplied

Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin-smart polymer conjugate.

A system has been developed for reversibly binding and thermoprecipitating biotinylated macromolecules. A high off-rate Ser45Ala (S45A) streptavidin mutant has been covalently conjugated to poly(N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer. The resulting conjugate is shown to coprecipitate biotinylated immunoglobulin G (IgG) and a biotinylated oligonucleotide in response to a thermal stimulus. Thermally precipitated biotinylated macromolecules can be released from the S45A-PNIPAAm conjugate by simple treatment with excess free biotin. This release step has been shown to be unique to the mutant streptavidin conjugate-a conjugate of wild type (WT) streptavidin and PNIPAAm does not release bound biotinylated molecules upon treatment with excess free biotin. The capture efficiency (fraction of target molecule precipitated from solution) of the S45A-PNIPAAm conjugate is similar to that of the WT-PNIPAAm conjugate for the biotinylated IgG target molecule (near 100%), but significantly smaller for the biotinylated oligonucleotide target (approximately 60% for the S45A-PNIPAAm conjugate compared to 80% for the WT-PNIPAAm conjugate). The release efficiency (fraction of originally precipitated target molecule released after treatment with free biotin) of the S45A-PNIPAAm conjugate is 70-80% for the biotinylated IgG target and nears 100% for the biotinylated oligonucleotide target. This system demonstrates the use of a high off-rate streptavidin mutant to add reversibility to a system based on smart-polymer-streptavidin conjugates.

Animals↗