%0 Journal Article
%T An Approach to the Production of Soluble Protein from a Fungal Gene Encoding an Aggregation-Prone Xylanase in Escherichia coli
%A Yilin Le
%A Jingjing Peng
%A Huawei Wu
%A Jianzhong Sun
%A Weilan Shao
%J PLOS ONE
%D 2012
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0018489
%X The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (¦Ò32). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by ¦Ò32. One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of ¦Ò32. The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018489