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Site-Specific Protein and Peptide Immobilization on a Biosensor Surface by Pulsed Native Chemical Ligation


During the last years native chemical ligation (NCL) gained in popularity as a method allowing the chemical synthesis of large peptides and entire proteins. NCL is particularly well-suited for chemoselective and nondenaturing attachment of biomolecules on solid substrates. In the present work, we show the feasibility of monitoring of custom peptide synthesis, NCL and its catalysis on silicon oxide modified gold surfaces by surface plasmon resonance (SPR). NCL of a model peptide-bradykinin thioester-was carried out and monitored with a custom-built SPR apparatus. Solid-phase produced bradykinin thioester was ligated to the surface in the presence of variable concentrations of 4-mercaptophenylacetic acid as transthioesterification catalyst. At catalyst concentration of 48 mM and above, the NCL reaction was maximal and identical to the reaction of the purified peptide-mercaptophenylacetic acid thioester. SPR curves indicate typical first-order kinetics with t(1/2) of 81 s for this aryl thioester, but of 104 min for the primary alkyl thioester. (c) 2007 Wiley Periodicals, Inc.
Microfluidic biosensor chips that are functionalized with cysteine derivatives are readily modified with peptides and proteins by pulsed native chemical ligation. The chemoselectivity ensures a homogeneous presentation of the biomolecule at the surface, and the ligand density can be tuned in a highly programmable way. The modified surfaces are selectively responsive towards complementary analytes, thus allowing accurate kinetic and thermodynamic information to be obtained.
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