P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

摘要: 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 effects several different C-terminal extension sequences on activity a set mutants. We show that two tail sequences, KNQHE (st5) H6KNQHE (st11), increase most mutants from 10- 20-fold and, some cases, result restoration biological cell. A third sequence, HHHHHH (st6), was not as effective increasing levels. All three functionally structurally silent, judged by lack DNA binding stability otherwise wild-type Arc. The properties st11 sequence make it an efficient system proteins, both because increased proteins can be rapidly purified using nickel-chelate affinity chromatography. containing EA28, RL31, SA32 mutations were background. thermodynamic EA28 (delta delta Gu approximately -0.4 kcal/mol) is reduced modestly compared parent, whereas RL31 -3.0 -3.3 substantially less stable.