Proteomics Application Notes

Proteomics Application Notes

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Introduction

Disaggregation and solubilization of protein aggregates in mild reagents is challenging. Most disaggregation protocols call for protein denaturation in harsh reagents such as detergents, concentrated guanidine-HCl, or 8M urea. Pressure can also be used to denature proteins, and high hydrostatic pressure has shown promise as a means of solubilizing and/or refolding insoluble aggregates due to its effects on electrostatic and hydrophobic interactions – two key components of aggregate formation [2-3]. Disaggregation by pressure works in manner similar to chemical disaggregation, with one significant advantage; pressure-disaggregated proteins do not require extensive clean-up to remove the high concentrations of denaturing chemicals required by conventional methods.Here we report that solubilization of aggregated β-Casein can be enhanced when carried out under high pressure, even in the absence of strong chaotropes. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced solubilization of β-Casein or similar aggregated proteins.

Introduction

Protein expression in E. coli is an efficient and commonly used method to generate large quantities of protein for research or therapeutic applications. Unfortunately, proteins expressed at high levels in E. coli are often packaged into inclusion bodies (IBs). These tightly-packed structures have the advantage of being composed of almost pure expressed protein, but the serious disadvantage that the protein is so tightly aggregated that high concentrations of chaotropes or detergents are required to extract soluble protein from the aggregates. These solubilization reagents must then be diluted or removed by buffer exchange, so that the extracted protein can be refolded into its native, functional conformation.

High hydrostatic pressure has shown promise as a means of disaggregating and solubilizing protein aggregates using relatively mild buffer conditions [1-4]. By disaggregating IBs without the high levels of denaturants required under conventional conditions, subsequent protein refolding can be improved.

Here we report that high hydrostatic pressure can be used to efficiently disaggregate proinsulin inclusion bodies in order to extract soluble proinsulin protein. This disaggregation can be carried out in mild buffer conditions at ambient temperature in as little as 5 minutes at 45kpsi.

Introduction
Pancreatic cancer has a very high mortality rate, primarily due to the fact that it is usually diagnosed at an advanced stage (Ranganathan 2009). Early diagnosis of this devastating disease could be crucial for improving treatment options and survival rates. The pancreas, as the site of insulin secretion, is also intimately linked to diabetes. A better understanding of the normal and diseased pancreatic proteome might give researchers better insights into pancreatic function and development in health and dysfunction in various disease states (Tonack et al., 2009; Chen et al., 2007).

Extraction of total proteins from tissue has generally been limited by the poor solubility of many proteins in traditional extraction buffers. This has been especially true for lipid-rich samples such as adipose tissue, but also for many other types of samples. Traditional detergent-based sample preparation methods may not adequately dissociate all proteins, especially hydrophobic proteins, which may be tightly associated with membrane lipids. Isolation of these proteins is often very inefficient, because the bulk of membrane proteins are often discarded in the insoluble fraction after extraction. As a result, proteomic analysis of tissues is often biased toward the more soluble proteins. We have previously described a method for efficient extraction of proteins from samples of a variety of mammalian tissues, using pressure cycling technology (PCT) and the novel chemistry of

Pressure BioSciences’ ProteoSolve-SB kit. Here we show that by using the new PCT MicroTubes, the ProteoSolve-SB protocol may be scaled down for use with tissue samples in the 10-20 mg size range. This scaled-down method is compatible with biopsy-size tissue samples.

Introduction

Pressure-enhanced proteolytic digestion exploits the ability of high hydrostatic pressure to promote protein denaturation and the access of proteolytic enzymes to their target sites. Pressure denaturation is fundamentally different from thermal denaturation as it occurs by virtue of hydration of hydrophobic

residues and by water saturation of protein substrate cavities normally inaccessible to solvent. Pressure denaturation is more efficient for hydrophobic proteins, while some hydrophilic soluble proteins are reported to retain relatively compact conformation, even when saturated by water molecules [1-6].