Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system

Abstract

To develop an efficient protein expression system, we designed a late embryogenesis abundant (LEA) peptide by mutating the LEA peptide constructed in our previous study (LEA-I). The peptide is based on the repeating units of an 11mer motif characteristic of LEA proteins from Polypedilum vanderplanki larvae. In the amino acid sequence of the 13mer LEA peptide, glycine at the 6th and 12th positions was replaced with other amino acids via point mutations. Glutamic acid, lysine, leucine, and asparagine in the LEA peptide at the 6th and 12th positions increased green fluorescence protein (GFP) expression. The GFP expression of the mutated LEA peptide was 1.5 to 2.0 times higher than that without LEA peptide. In contrast, the serine-containing mutated LEA peptide has low GFP expression levels. We hypothesize that the position of amino acids and the nature of amino acid in LEA peptide are important for our coexpression system. These data suggest that the size, structure, and charge of amino acids in the LEA peptide improve the protection and expression of the target protein. The amino acid balance also plays an important role in the expression of the target protein.

Intermittent induction of LEA peptide by lactose enhances the expression of insecticidal proteins in Bacillus thuringiensis

“…Point mutations, size, and structure of amino acids affect the function of LEA peptides. From our previous study [ 28 ], it is already established that point mutations play a vital role in the enhancement of the function of LEA peptides in the coexpression of targeted proteins in cells. In the amino acid sequence of the 13‐mer LEA peptides, glycine was replaced with glutamic acid at the position of amino acids 6 and 12 in the LEA‐E peptide, and glycine was replaced with lysine at the position of amino acids 6 and 12 in the LEA‐K peptide.…”

“…In the LEA peptide coexpression, the importance of the size and position of amino acids that play a vital role in the coexpression system have been already reported by Pathak et al . [ 28 ]. Finally, the most important feature of the Bt‐LEA‐E transformant is its induction by intermittent induction of lactose.…”

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides

“…G3LEA proteins such as LEA-I ( 23 ) and LEA-K peptides ( 24 ) were used for coexpression with Ab3 lipase to improve Ab3 lipase protein expression. A few reports have demonstrated that the presence of G3LEA proteins can provide efficient protein expression due to their function in “molecular shielding” ( 22 – 24 ).…”

“…Most of the G3LEA proteins have been reported to serve a protective role for plants and animals during the onset of water stress conditions ( 15 – 21 ). Recently, a novel approach of involving G3LEA protein in coexpression with target protein within its host, Escherichia coli , enhanced the protein solubility drastically, leading to better production of functional protein ( 22 – 24 ). Unlike others, the method is simple and efficient by only utilizing the conserved 11 amino acid residues of G3LEA protein ( 23 ).…”

“…Unlike others, the method is simple and efficient by only utilizing the conserved 11 amino acid residues of G3LEA protein ( 23 ). Improvement of this method is also notable as it only manipulates hydrophobic, charged amino acids among 11 amino acid residues of G3LEA protein via point mutations ( 24 ). Since the coexpressed peptide is small, the impact toward the functional protein is insignificant, and this allows simple, convenient protein purification ( 23 ).…”