An Approach to the Production of Soluble Protein from a Fungal Gene Encoding an Aggregation-Prone Xylanase in Escherichia coli

Abstract

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 (σ32). 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 σ32. 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 σ32. 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.

Tat Peptide-Mediated Soluble Expression of the Membrane Protein LSECtin-CRD in Escherichia coli

“…The TEM ultramicrotomy as a routine technique has been widely applied in identifying of the inclusion body location in bacterial cells [24,[26][27][28][29]. In this study, it was used to elucidate the subcellular structure changes of bacterial cells after overexpressed Tat tag and Tat free protein.…”

Enhanced intracellular soluble production of 3‐ketosteroid‐Δ1‐dehydrogenase from Mycobacterium neoaurum in Escherichia coli and its application in the androst‐1,4‐diene‐3,17‐dione production

“…In order to enhance the soluble expression level of recombinant proteins, the formation of IBs should be reduced. Currently, the strategies used for reducing the formation of IBs are as follows: (1) lowering the growth temperature and concentration of the inducer; (2) enabling secretion of recombinant proteins into the periplasm; (3) co‐expressing chaperone genes; and (4) increasing the concentration of viscous osmolytes …”

Heterologous expression, purification, crystallization and preliminary X-ray analysis ofTrichoderma reeseixylanase II and four variants

“…reesei cells express Xyn II with a leader peptide that is cleaved during secretion, producing a 21 kDa protein with an N-terminal pyroglutamate residue. Previously, this protein has been expressed in E. coli fused to a 36-amino-acid tag at the N-terminus (He et al, 2009) or an N-terminal periplasmic signal peptide (Le et al, 2011). We designed new expression vectors to produce high levels of proteins without N-terminal extensions for crystallography.…”