Premium
P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences
Author(s) -
Milla Marcos E.,
Brown Bronwen M.,
Sauer Robert T.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560021219
Subject(s) - mutant , repressor , microbiology and biotechnology , biology , proteolysis , arc (geometry) , mutant protein , biochemistry , gene , gene expression , enzyme , geometry , mathematics
Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli , which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C‐terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H 6 KNQHE (st11), increase the expression levels of most mutants from 10‐ to 20‐fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, (st6), was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild‐type Arc. The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel‐chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background. The thermodynamic stability of the EA28 mutant (ΔΔ G u ‐0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (ΔΔ G u ‐3.0 kcal/mol) and SA32 mutant (ΔΔ G u ‐3.3 kcal/mol) are substantially less stable.