DRUG DISCOVERY INTERFACEExtreme sensitivity of botulinum neurotoxin domains towards mild agitation
Section snippets
INTRODUCTION
Understanding and monitoring the stability of proteins are important factors in their therapeutic applications. Botulinum neurotoxins (BoNTs) are most lethal of all toxins.1,2 The neurotoxins and their nontoxic fragments are also finding increasing application as therapeutic and prophylactic agents,3, 4, 5, 6, 7, 8, 9 and are targets of drug development.10, 11, 12, 13 These neurotoxins are initially expressed by strains of Clostridium botulinum as 150-kDa single polypeptides along with other
BoNT/A LC, Chemicals, Buffers, and Reagents
The 449-residue recombinant BoNT/A LC (LcA) with an extra valine residue at position 218 was expressed and purified as described.17 The homogeneous preparation was stored at −20°C in 50 mM Na-phosphate, pH 6.5 containing 150–250 mM NaCl and 2 mM EDTA. Recombinant LcB was purified as published,23 those of LcD and LcG will be published elsewhere. Recombinant LcE was purchased from Bibitech (Dartmouth, MA). Recombinant Hc proteins were purified in Dr. Smith's laboratory. Before use, each 1 mL of
Denaturation of LcA Induced by Mechanical Stirring
Agitation by stirring of 2.7 mL solution in all experiments was set at a speed that did not produce any vortex or visible turbulence at the surface, which was 13 mm above the “Star Head” magnetic stirrer (or 18 mm above the “Micro-Flea” stirrer bar) and 5 mm above the center of light path. The stirring therefore will be considered as mild. Figure 2 (curve 1) shows that mild stirring of LcA with the “Star Head” bar led to rapid loss of its CD signal related to α-helical secondary structure as a
Extreme Instability
Protein denaturation by shaking or mechanical agitation is an old observation. As early as 1927, Wu30 (and the references therein) reported shaking a solution of protein destabilizes or denatures protein, forming aggregates and precipitates which are often catalytically inactive. Yet, destabilization leading to aggregation still remains a common problem with protein pharmaceuticals.31,32 These destabilizations are usually attributed to exposure of kinetically stable unfolded33 protein molecules
SCHEME I
The CD technique employed in these studies could not detect any intermediate such as TS* or its aggregate-competent D1 (Fig. 2B) but agitation might have simply enhanced the formation of D1 that rapidly formed the inactive species Dm and Dm+1. In the absence of significant agitation, the catalytically inactive or poorly active D1 may have accumulated in a solution yielding suboptimal catalytic activity of a LC preparation. Indeed, large variations in the catalytic activity of the same LcA are
Acknowledgements
The research described herein was sponsored by JSTO-CBD (RIID 3.10011_06_RD_B (to SAA)). We thank Dr. John Carra, Dr. Wieslaw Swietnicki, and Dr. Frank Lebeda for helpful comments and critical reading of the manuscript.
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Cited by (17)
The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site
2013, Journal of Biological ChemistryCitation Excerpt :First, LcA with a high molecular mass of 51 kDa yields only an ∼20 μm solution at 1 mg/ml, a relatively high concentration that often leads to its own precipitation. Second, titration in ITC uses constant stirring that leads to LcA precipitation (38) and a syringe, the metallic part of which most likely induces autocatalytic degradation (23, 25, 39). Therefore, we chose its C-terminal peptide LcA-1 in place of the full-length LcA.
Forced degradation of therapeutic proteins
2012, Journal of Pharmaceutical SciencesCitation Excerpt :Mechanical stress testing is often performed to evaluate the stabilizing properties of excipients, such as surfactants added to formulations with the intention of reducing agitation- and surface-induced aggregation.14,90 Mechanical stress testing can be performed as agitation (shaking)14,90–97 or stirring studies,94,95,98–101 but also pumping,5,102–104 vortexing,105–107 sonication,108,109 and special shearing devices110–114 have been used for this purpose. During mechanical stress testing, proteins experience several types of stress factors, such as shearing, exposure to liquid–air and liquid–container interfaces, cavitation, and local warming effects,18 which can result in physical and chemical instability.
Basic tetrapeptides as potent intracellular inhibitors of type a botulinum neurotoxin protease activity
2011, Journal of Biological ChemistryCitation Excerpt :Recently, Burnett et al. (5) reported determination of affinity constant Ka for BoNT/A LcA by a pseudopeptide using isothermal titration microcalorimetry. This technique was not used for confirmation of the studies described here because of the inherent instability of BoNT domains toward mild agitation (37) that is necessary in the microcalorimetry technique. In addition, the metallic needles in such instruments are likely to catalyze an autocatalytic fragmentation reaction of BoNT/A LcA (21, 38) that would almost certainly affect the Ka determination.
Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery
2010, Protein Expression and PurificationRapid product analysis and increased sensitivity for quantitative determinations of botulinum neurotoxin proteolytic activity
2010, Analytical BiochemistryCitation Excerpt :Because all BoNT domains, including LcA and LcD, are prone to surface denaturation [26], the observed stimulating effects on LcA and LcD activity may suggest that the detergents preserve the most active conformation of LcA that was reflected in the activity stimulation. In fact, protection of LcA from precipitation due to surface denaturation was recently demonstrated [26]. Except for TGT-7, variations of the effect within Tergitols are not significant.