Enhancing recombinant protein quality and yield by protein stability profiling.

The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.

Protein engineering of the colony-stimulating factor-1 receptor kinase domain for structural studies.

A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.

Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration.

The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 degrees C, from 40 to 62 degrees C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH4+>Na+>Li+>K+. Of the divalent cations tested: Mg+2 and Mn+2 significantly stabilize the protein, while Ni+2 dramatically destabilizes the protein. Stability measurements performed in combined Mg+2 plus Na+ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein.

Dihydroquinone ansamycins: toward resolving the conflict between low in vitro affinity and high cellular potency of geldanamycin derivatives.

Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.