Immobilization methods for the rapid total chemical synthesis of proteins on microtiter plates

The chemical synthesis of proteins typically involves the solid‐phase peptide synthesis of unprotected peptide fragments that are stitched together in solution by native chemical ligation (NCL). The process is slow, and throughput is limited because of the need for repeated high performance liquid chromatography purification steps after both solid‐phase peptide synthesis and NCL. With an aim to provide faster access to functional proteins and to accelerate the functional analysis of synthetic proteins by parallelization, we developed a method for the high performance liquid chromatography‐free synthesis of proteins on the surface of microtiter plates. The method relies on solid‐phase synthesis of unprotected peptide fragments, immobilization of the C‐terminal fragment and on‐surface NCL with an unprotected peptide thioester in crude form. Herein, we describe the development of a suitable immobilization chemistry. We compared (i) formation of nickel(II)‐oligohistidine complexes, (ii) Cu‐based [2 + 3] alkine–azide cycloaddition and (iii) hydrazone ligation. The comparative study identified the hydrazone ligation as most suitable. The sequence of immobilization via hydrazone ligation, on‐surface NCL and radical desulfurization furnished the targeted SH3 domains in near quantitative yield. The synthetic proteins were functional as demonstrated by an on‐surface fluorescence‐based saturation binding analysis. Copyright © 2017 European Peptide Society and ...
Source: Journal of Peptide Science - Category: Biochemistry Authors: Tags: Special Issue Article Source Type: research