Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2).

Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

Peptide inhibitors of human HMG-CoA reductase as potential hypocholesterolemia agents.

Hypercholesterolemia may lead to obesity and cardiovascular diseases. To prevent hypercholesterolemia, many drugs have been developed while searching for better drugs to treat hypercholesterolemia has never been ended. Other than small molecule drugs, peptide drugs are gaining more visibilities in many therapeutic areas. In the present study, we employed phage-display techniques to screen peptide inhibitors against human HMG-CoA reductase. The results indicate that the tetrapeptide PMAS inhibits hHMGR effectively (IC50=68 µM), could be a lead compound to develop hypocholesterolemic agents.

Kinetics study on the HIV-1 ectodomain protein quaternary structure formation reveals coupling of chain folding and self-assembly in the refolding cascade.

Entry of HIV-1 into the target cell is mediated by the envelope glycoprotein consisting of noncovalently associated surface subunit gp120 and transmembrane subunit gp41. To form a functional gp41 complex, the protein undergoes hairpin formation and self-assembly. The fusion event can be inhibited by gp41-derived peptides at nanomolar concentration and is highly dependent on the time of addition, implying a role of folding kinetics on the inhibitory action. Oligomerization of the gp41 ectodomain was demonstrated by light scattering measurements. Kinetic study by stopped-flow fluorescence and absorption measurements (i) revealed a multistate folding pathway and stable intermediates; (ii) showed a dissection of fast and slow components for early and late stages of folding, respectively, with 3 orders of magnitude difference in the time scale; (iii) showed the slow process was attributed to misfolding and unzipping of the hairpin; and (iv) showed retardation of the native hairpin formation is assumed to lead to coupling of the correctly registered hairpin and self-assembly. This coupling allows the deduction on the time scale of intrachain folding (0.1-1 s) for the protein. The folding reaction was illustrated by a free energy profile to explain the temporal dichotomy of fast and slow steps of folding as well as effective inhibition by gp41-derived peptide.

A novel method for preparing a protein-encapsulated bioaerogel: using a red fluorescent protein as a model.

A recombinant red fluorescent protein, DsRed, was chosen as a model protein to prepare a protein-encapsulated bioaerogel, DsRed-SAG. It was prepared using sol-gel polymerization of tetraethyl orthosilicate (TEOS) with an ionic liquid as the solvent and pore-forming agent. The DsRed-SAG bioaerogel was characterized by Fourier transformation infrared, scanning electron microscopy and Brunauer-Emmett-Teller measurements. It was found that the as-prepared bioaerogel had high porosity, and the silica network exhibited little shrinkage during the drying process. The stability of the bioaerogel was monitored by fluorescence spectroscopy and confirmed by confocal laser scanning microscopy. In addition, the protection of the encapsulated proteins by the silica network was further investigated using the degradation test by a protease. The results indicated that the as-prepared protein was quite stable during formation of the protein-containing wet gel and extraction of the ionic liquid, demonstrating that the new method can be extended to prepare other protein-encapsulated bioaerogels.