Selecting Heperase Micromolecule Peptide Inhibitor From A Phage Display Peptide Library

Cancer's fatality not only lies in its malignant proliferation, but also its metastasis.Heparanase is a key enzyme which degradate the extracellular matrix and basementmembrane resulting in tumor cells metastasis extensively in vovo. Thus heparanase isthe target of many inhibitors.Heparanase is an endoglycosidase which cleaves internal glycosidic bonds ofheparan sulfate of the heparin sulphate proteoglycan in the extracellular matrix andbasement membrane. In the mid 1980s, it was firstly discovered that heparanasecorrelates with tumor metastasis, however, its development was held back because ofunstable nature, lack of simple and quick detection methods and technical difficulty ofpurification. Fortunately, quite a few laboratories have cloned HPA genes separately,which developed a new and potential field of tumor metastasis research.The gene of heparanase N-terminus subunit was amplified by PCR and cloned itinto the pET-24a(+) vector. According to the results of SDS-PAGE analysis,heparanase N-terminus subunit realized its expression in E.coli. After purification anddislysis, the concentration of goal protein reach 1.9 mg/mL and the purity was over98ï¼…. Heparanase N-terminus subunit was used as the target for biopanning from aPh.D.-C7CTM Phage Display Peptide Library to obtain short peptides which bindspecifically to heparanase N-terminus subunit. After 5 rounds of screening, thepositive phage clones were identified by phage enzyme linked immunosorbent assay(phage ELISA), which shows the positive phage monoclone bind heparanaseN-terminus subunit specifically, however, not bind coating solution and humanepidermal growth factor (hEGF). 17 amino acid sequences were deduced by 25 DNAsequencing, but no conserved motif was found in these peptides. Meanwhile, PeptideP1 was the consensus amino acid sequence of biopanning. Then Peptide P1 andPeptide P8 were chemically synthesized in Xi'an Langene Bioscience Company.Human breast tumor MCF-7 in which Heparanase N-terminus subunit is notexpressed was used as negative control, human hepatoma HepG-2 as positive control, a Transwell invasion model in vitro was established to detect the effect of Peptide P1and Peptide P8 on invasion capacities of HepG-2 cells in vitro. The results showedthat Peptide P1 could inhibit the invasion capacities of HepG-2 cells in vitro, whilePeptide P8 couldn't. The invasion inhibition ratio reached 57.1ï¼….