Biosynthesis and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori

We aimed to investigate the self-organization/self-assembly mechanisms of silkworm fibroin-based material. In the present study, for the first time, we designed and multimerized four DNA monomer sequences from structurally simple fibroin crystalline peptides or analog, [GAGAGX] (X = A, S, Y and V) to encode polypeptides [GAGAGX](16) (eGA, eGS, eGY and eGV) using a head-to-tail construction strategy. Multimers were cloned into pGEX-KG and fusion proteins GST-[GAGAGX](16) (KGA, KGS, KGY and KGV) were efficiently expressed in Escherichia coli. These fusion proteins were isolated and purified by GST affinity chromatography and confirmed by SDS-PAGE and Western blot analysis using antibody reactive to GST The polypeptides were cleavaged from GST fusion proteins by digesting with thrombin enzyme. The composition of the four polypeptides was confirmed by composition analysis of amino acids, and their abilities to form beta-sheet structure were determined by ThT fluorescence Spectral analysis. The content of beta-sheet among the four polypeptides followed the order: eGS>eGV>eGY>eGA. (C) 2008 Elsevier B.V. All rights reserved.