해양유래 홍합실크단백질의 대장균에서의 발현연구

Abstract

Silk has been used as a novel texture for centuries. Nowadays, the usage of silk reaches to the material industry and medical area. Even though the silk protein has great potentials due to its extraordinary strength and toughness, it has been hard to develop successful techniques for mass production of silks. Until now, most silk studies have been focused on the spider and silkworm. However, by investigating new silk-bearing organisms, silks with new properties can be found and this attempt can be a solution for practical production of silk protein. In the present work, we tried to express recombinant mussel silk protein in Escherichia coli. Firstly, we compared the silk genes from spider, silkworm, and mussel. Notably, we observed that the mussel silk gene consists of many repeats with abundance of glycine and alanine. In the same way, elastic proteins which sequence are similar to silk have plenty of polymer repeats with non-repeated region. Accordingly, we designed mussel silk gene which contains alternating repeated (hydrophobic) and non-repeated regions. After chemical synthesis of mussel silk gene, we constructed the recombinant vector based on the pET23 vector containing strong T7 promoter. However the expression level was too low, thus we tried other vectors, pCOLD DNA and pQE30. These alterations were for use of co-expression of cold shock protein and weaker promoter (T5 promoter). In addition, other strategies such as the expressions of partial mussel silkprotein, diverse culture conditions, different protein detection methods, and several reagent treatments were also tried. Nevertheless, the tryouts could not bring considerable changes on the expression level of the mussel silk protein. Next, we tried to make fusion silk protein with GST or baculoviral polyhedrin protein. Interestingly, in the case of cold shock protein and GST fusion protein systems, the retardation of cell growth was prominent. This phenomenon might be considered that a soluble-form target protein has significant burden on E.coli. Since baculoviral polyhedrin has been known to induce expression of target protein as an insoluble form, we could express the mussel silk proteins as insoluble form with high cell growth.