Conformation and stability of barley chymotrypsin inhibitor-2 (CI-2) mutants containing multiple lysine substitutions.


Abstract

A major goal of agricultural biotechnology is to increase the nutritional value of maize seed through the expression of heterologous proteins enriched in lysine. One promising candidate is barley chymotrypsin inhibitor-2 (CI-2), a plant protein that has been extensively characterized with respect to structure and function. Based on the tertiary structure of wild-type (WT) CI-2, five mutants with lysine contents ranging from 20 to 25 mol percent were designed, expressed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Inasmuch as previous transgenic experiments suggested that proper folding and stability may be essential for in vivo accumulation of the engineered proteins in plant cells, we first undertook an in vitro study of the conformation and thermodynamic stability of the CI-2 mutants in order to select an ideal candidate for plant expression. Mutant and WT CI-2 proteins had similar circular dichroism spectra, suggesting similar secondary structures. However, differences in the accessibility of the sole tryptophan residue, Trp24, indicated that the local conformation differed among the mutants. The thermodynamic stability of the mutants ranged from <2 to 4.9 kcal/mol compared with approximately 7 kcal/mol for the wild-type protein. In conjunction with proteolytic stability studies, we have identified one mutant that has the potential to be expressed in a stable manner in plant cells. Study holds ProTherm entries: 7045 Extra Details: barley chymotrypsin inhibitor-2; nutritional enhancement;,protein conformation; protein engineering; protein stability

Submission Details

ID: fxW86Kmf3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:33 p.m.

Version: 1

Publication Details
Roesler KR;Rao AG,Protein Eng. (1999) Conformation and stability of barley chymotrypsin inhibitor-2 (CI-2) mutants containing multiple lysine substitutions. PMID:10585502
Additional Information

Sequence Assay Result Units