An efficient procedure for the selection of high affinity clones from a heptapeptide phage display library was developed. Lysozyme was used as a model protein to demonstrate the selection strategy. Effect of bovine serum albumin (BSA) concentration on screening the phage library was discussed and proper BSA concentration on plate blocking was determined. The elution procedure was improved by alternatingly eluting the bound phages with glycine-HCl buffer (pH 2.2) and high-concentration target protein solution. The modified method was compared with others including the conventional protocol, and the results confirmed that the modified procedure could yield high affinity phages that might be lost by other screening methods. Through comparison of the DNA sequences of foreign peptides of the clones showing specificity to lysozyme molecules, the HWWW motif was found to be the necessary amino acid sequence for the affinity. The electrostatic and hydrophobic interactions are considered to contribute to the affinity for the protein. Moreover, protein chromatography with the immobilized HWWWPAS on Sepharose gel indicated the strong binding affinity of the peptide for lysozyme.

Interest in producing large quantities of plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Hydrophobic chromatography is a popular technique in the downstream processing of plasmid DNA. However, the low capacity and the high mass transfer resistance of most commercially available packings for bio-macromolecules limit their application in a large-scale process. In this work, a hydrophobic absorbent with wide pores was synthesized by the solid porogenic method. Analyses by scanning electron microscopy and mercury intrusion porosimetry revealed that the matrix contained two families of pores, i.e., micropores smaller than 100 nm and superpores of 500-7300 nm. The superpores provided not only convective flow channels for the mobile phase, but also a large surface for biomolecules binding. So the chromatographic process can be operated at high flow rate with high column efficiency and low backpressure as identified on a 2-mL column (5mm i.d., 2cm length). With a loading up to 2.6 mg of 5.4 kb plasmid (pcDNA3) in 8mL feedstock and operated at a flow rate as high as 20 cm/min, nearly 100% of plasmid was recovered with a purity of 100%. The results indicate that the hydrophobic medium is promising for high-speed purification of plasmid DNA.

Lysozyme adsorption and purification by expanded bed chromatography of a customized Nd-Fe-B alloy-densified agarose (NFBA) gel modified with Cibacron Blue 3GA (CB) were investigated, and the results were compared with those obtained with CB-modified Streamline gel (CB-Streamline). The NFBA gel had a mean size of 102m and a mean density of 1.88g/ml. The breakthrough behavior of lysozyme was modeled considering the dispersion of the liquid and solid phases and the diffusive mass transport of protein to the solid phase. Using independently determined parameters, the model prediction agreed reasonably well to the experimental data. Although the two dye-ligand adsorbents showed nearly the same static capacity, the dynamic binding capacity of the CB-NFBA gel was nearly twice that of the CB-Streamline gel. Moreover, lysozyme was purified from chicken egg white solution by the expanded beds with the two adsorbents, and the expanded bed with the CB-NFBA gel produced much larger purification factor than that with the CB-Streamline gel.All the results indicated that the small-sized dense medium CB-NFBA gel was more efficient as an expanded bed adsorbent.

The refolding kinetic behavior of denatured-reduced lysozyme in the presence of folding aids (acetamide, acetone, thiourea, l-arginine or glycerol) was studied utilizing a simplified model describing the competition between first-order folding reaction and third-order aggregation. It was found that the protein folding aids could be categorized into two groups. One of them at proper concentrations, such as acetamide, acetone, thiourea and l-arginine, stabilized unfolded protein or folding intermediates. In the presence of these additives, the folding rate decreased with increasing their concentration, and there existed a concentration where the aggregation rate constant was minimized. So, there was an optimum concentration for the folding aids to produce a high yield. The other group was protein stabilizers such as glycerol. In the presence of this kind of folding aids, both the refolding rate and yield were enhanced by increasing their concentration to a proper value. Moreover, their effect on improving protein refolding was additive to those of the first group. So the cooperative application of the two kinds of folding aids could result in favorable refolding rate and yield of protein.

Sorbitan trioleate (Span 85) modified with Cibacron Blue F-3GA (CB) was used as an affinity surfactant (CB-Span 85) to form affinity-based reversed micelles in n-hexane. It was found that the addition of hexanol to the reversed micellar system resulted in a significant increase in water content and hydrodynamic radius of the affinity-based reversed micelles. Moreover, the reversed micelles with hexanol revealed broader aggregation number distribution and larger average aggregation number than the reversed micelles without hexanol addition. This is considered to be due to the decreases in the micellar curvature and rigidity of the micellar interfacial layer and the increase in the micellar interfacial fluidity. Consequently, the solubilization capacity of lysozyme increased about 70% in the reversed micellar solution with 3 vol% hexanol. On the other hand, the capacity of BSA was only 30% increased under the same conditions due to its larger molecular size than lysozyme. Kinetic analysis revealed that the increase in the micellar interfacial fluidity in the presence of hexanol resulted in faster release of lysozyme from the micelles, thus leading to an increase of the overall volumetric mass transfer coefficient in the back extraction.