title: An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli.
abstract: Bacterial cells can be engineered to express non-native genes, resulting in the production of, recombinant proteins, which have various biotechnological and pharmaceutical applications. In eukaryotes, such as yeast or mammalian cells, which have large genomes, a higher recombinant protein expression can be troublesome. Comparatively, in the Escherichia coli (E. coli) expression system, although the expression is induced with isopropyl β-d-1-thiogalactopyranoside (IPTG), studies have shown low expression levels of proteins. Irrespective of the purpose of protein production, the production process requires the accomplishment of three individual factors: expression, solubilization and purification. Although several efforts, including changing the host, vector, culture parameters of the recombinant host strain, co-expression of other genes and changing of the gene sequences, have been directed towards enhancing recombinant protein expression, the protein expression is still considered as a significant limiting step. Our protocol explains a simple method to enhance the recombinant protein expression that we have optimized using several unrelated proteins. It works with both T5 and T7 promoters. This protocol can be used to enhance the expressions of most of the proteins. The advantages of this technique are presented below:•It produces several fold increase in the expression of poorly expressed, less expressed or non-expressed recombinant proteins.•It does not employ any additional component such as chaperones, heat shock proteins or co-expression of other genes.•In addition to being inexpensive, easy to manage, universal, and quick to perform, the proposed method does not require any commercial kits and, can be used for various recombinant proteins expressed in the E. coli expression system.
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793 022, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Chhetri
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793 022, India.
authors.forename: Parismita
authors.initials: P
authors.lastname: Kalita
authors.affiliation: Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793 022, India.
authors.forename: Timir
authors.initials: T
authors.lastname: Tripathi
keywords: E. coli; Ethanol; Expression of inducible proteins; High-level protein expression using ethanol; Osmotic stress; Protein expression; Protein induction; Recombinant protein