Lianfen Zhang

Expression, purification, and characterization of recombinant human serum albumin fusion protein with two human glucagon-like peptide-1 mutants in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) is a 30-residue peptide hormone secreted by intestinal L-cells in response to nutrient ingestion. In the present study, overlapping PCR technology was employed to construct two GLP-1 mutants (GLP-1(A2G))2 and human albumin (HSA) genes in vitro without linker. The spliced gene, (GLP-1(A2G))2-HSA, was over expressed under the control of promoter AOX1 and Mat alpha signal peptide in Pichia pastoris. SDS-PAGE and Western blotting were applied to assay the recombinant fusion protein in the culture broth. The results demonstrated that the recombinant (GLP-1(A2G))2-HSA concentration in the broth could reach a level of 245.0 mg/L and the expressed fusion protein was capable of cross-reacting with anti-human GLP-1 and anti-human albumin antibody. The recombinant (GLP-1(A2G))2-HSA protein was purified by ultrafiltration, columns of Q-sepharose fast flow and Superdex 75 size-exclusion. The recombinant (GLP-1(A2G))2-HSA protein obtained could lower in vivo glucose concentration in blood and stimulate in vitro islet cell proliferation. In mouse model, the fusion protein was detectable in plasma even 308 h after a single subcutaneous dose of 1.25 mg/kg. The result showed that the terminal biological half-time of the protein was about 54.2 h which is 650-fold longer than that of GLP-1. The pharmacokinetic analysis of the protein suggests its promising application in clinical medicine.

Anti-angiogenic activity of julibroside J8, a natural product isolated from Albizia julibrissin

PURPOSE The purpose of this study was to investigate the anti-angiogenic properties of julibroside J(8), a triterpenoid saponin isolated from Albizia julibrissin. METHODS In the presence of juliborside J(8,) the growth of human microvascular endothelial cells (HMEC-1), four human tumor cell lines, and a normal cell line (MRC-5) was evaluated by MTT assay. The in vivo anti-angiogenic effect of julibroside J(8) was evaluated on a chorioallantoic membrane (CAM) and in transplanted colon carcinoma cells in a nude mice neovascularisation model. RESULTS Treatment with 0.5-4 microg/ml julibroside J(8) resulted in dose-dependent inhibition of growth, migration, and tube formation in HMEC-1 cells; julibroside J(8) also inhibited the formation of microvessels on CAM at a concentration of 10-50 microg/egg and reduced vessel density within tumor at a concentration of 0.5-3mg/kg. CONCLUSIONS Julibroside J(8) may be a potent anti-angiogenetic and cytotoxic drug; further investigation is warranted.

Identification of binding peptides of the ADAM15 disintegrin domain using phage display.

ADAM15 plays an important role in tumour development by interacting with integrins. In this study, we investigated the target peptides of the ADAM15 disintegrin domain. First, we successfully produced the recombinant human ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of subtractive selection with streptavidin. By using the BLAST software and a relevant protein database, integrin ανβ3 was found to be homologous to peptide A. Synthetic peptide A had a highly inhibitory effect on RADD-integrin αvβ3 binding. The results demonstrate the potential application of short peptides for disrupting high-affinity ADAM-integrin interactions.