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

Abstract

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,KNQHE (st1 I), 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, H H H H H H (st@, 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 st1 1 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 st1 1 background. The thermodynamic stability of the EA28 mutant (AAG, = -0.4 kcal/mol) is reduced modestly compared to the st1 1 parent, whereas the RL31 mutant (AAG, = -3.0 kcal/mol) and SA32 mutant (AAG, = -3.3 kcal/mol) are substantially less stable.

A polymetamorphic protein

“…63 The arc-st11 gene includes the coding sequence for the st11 C-terminal extension (H 6 KNQHE) to stabilize the proteins against degradation during expression and to allow for affinity purification. 64 Wild-type Arc and S-VLV proteins were overexpressed in Escherichia coli strain BL21(kDE3) cells transformed with the appropriate variant of pET800, and purified by Ni 2þ -NTA affinity chromatography under denaturing conditions by loading cleared cell lysates in 100 mM NaH 2 PO 4 (pH 8.0), 10 mM Tris, 6M guanidine hydrochloride, 10 mM imidazole onto 3 mL Ni 2þ -NTA resin per liter of culture, washing with 40, 40, and 20 mL fractions of the same lysis/loading buffer, and eluting with low pH using 6M guanidine hydrochloride containing 0.2M acetic acid. After elution, proteins were refolded by dialysis into SB250 buffer [50 mM Tris (pH 7.5), 250 mM KCl, 0.2 mM EDTA] for 48 h at 4 C, with one buffer change after 24 h.…”

Biochemical characterization of recombinant polypeptides correspondingto the predicted ²±± fold in Aux/IAA proteins

“…It may be argued that the addition of the C-terminal (His)6 tag is responsible for the association behavior of the expressed [3etet peptides. However, C-terminally (His)6-tagged recombinant Arc repressor protein, which is of similar size (64 amino acids) and predicted secondary structure as the (His)6-tagged [3etc~ peptides, has virtually identical association properties and CD spectra as wild type Arc protein [25]. Thus, it is unlikely that the C-terminal (His)6 tag significantly affects the biochemical properties of the [3c~c~ peptides characterized in our study.…”

Transcriptional repressor CopR: dissection of stabilizing motifs within the C terminus

“…Previously, we had also shown that mutated protein Cop∆5, with a half-life of 31p6 min, is moderately less stable than wild-type CopR. Four of the C-terminal 7 aa and two of the C-terminal 5 aa are hydrophilic, which does support the role of hydrophilic amino acids in protecting more hydrophobic regions of the protein against proteolytic attack, as suggested by the publications of R. T. Sauer's group (Milla et al, 1993 ;Parsell & Sauer, 1989 ;Parsell et al, 1990). …”

“…Furthermore, residues closer to the C terminus appeared to play a greater role in determining the proteolytic susceptibility of the proteins than those farther away (Parsell et al, 1990). In all cases, however, the extensions were found to be unstructured and did not interact with the folded portions of the protein (Milla et al, 1993), and the stabilizing function was explained simply with the protection of hydrophobic parts of the proteins by these unstructured hydrophilic\ polar residues.…”

“…Pioneering work in this field has been done by the group of R. T. Sauer, who investigated sequences stabilizing the Arc repressor and the unstable N terminus of λ repressor (e.g. Bowie & Sauer, 1989 ;Parsell & Sauer, 1989 ;Parsell et al, 1990 ;Milla et al, 1993Milla et al, , 1994. They found that hydrophilic and polar residues within C-terminal extensions can stabilize otherwise unstable proteins, and even classified amino acids depending on their stabilizing effect.…”