Overview of Approaches to Preventing and Avoiding Proteolysis During Expression and Purification of Proteins

Proteases are enzymes that cleave proteins. They occur widely in nature and serve a fundamental role in cellular homeostasis; however, their presence can result in unwanted protein degradation during recombinant protein expression and purification. This unit introduces proteases, specifically outlining the types commonly encountered during production of recombinant proteins. The strategies used to avoid and to prevent proteolysis are also highlighted with extensive consideration of the molecular, technical, and logistical methodologies involve

Avoiding Proteolysis During Protein Purification

All cells contain proteases which hydrolyze the peptide bonds between amino acids in a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach; firstly inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographical purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. Here, commonly used approaches to reducing or avoiding proteolysis during protein purification and subsequent chromatography are reviewed

Influence of Cultivation Conditions on the Production of a Thermostable Extracellular Lipase from Amycolatopsis Mediterranei DSM 43304

Among several lipase producing actinomycete strains screened, Amycolatopsis mediterranei DSM 43304 was found to produce a thermostable, extracellular lipase. Culture condition and nutrient source modification studies involving carbon sources, nitrogen sources, incubation temperature and medium pH were carried out. Lipase activity of 1.37 ± 0.103 IU/ml of culture medium was obtained in 96 h at 28 °C and pH 7.5 using linseed oil and fructose as carbon sources and a combination of phytone peptone and yeast extract (5:1) as nitrogen sources. In optimal culture conditions the lipase activity was enhanced 12-fold with a 2-fold increase in lipase specific activity. The lipase showed maximum activity at 60 °C and pH 8.0. The enzyme was stable between pH 5.0–9.0 and temperatures up to 60 °C. Lipase activity was significantly enhanced by Fe3+ and strongly inhibited by Hg2+. Li+, Mg 2+ and PMSF significantly reduced lipase activity, whereas other metal ions had no significant effect at 0.01 M concentration. A. mediterranei DSM 43304 lipase exhibited remarkable stability in the presence of a wide range of organic solvents at 25% (v/v) concentration for 24h. These features render this novel lipase attractive for potential biotechnological applications in organic synthesis reactions

In Vitro Antimicrobial Activity and Mechanism of Action of Novel Carbohydrate Fatty Acid Derivatives Against Staphylococcus Aureus and MRSA

Aims: This study investigates the antimicrobial activity and mode of action of novel carbohydrate fatty acid (CFA) derivatives against Staphylococcus aureus and methicillin-resistant Staph. aureus (MRSA). Methods and Results: Minimum inhibitory concentrations (MIC) and the effect of CFA derivatives on lag phase were determined using a broth microdilution method. Lauric acid carbohydrate esters and corresponding ether analogs showed the greatest antimicrobial activity with MIC values between 0.04 to 0.16mmol l-1. Leakage studies at 260nm following exposure to CFA derivatives at 4X MIC showed a significant increase in membrane permeability for all compounds, after ca. 15 minutes exposure except for the lauric beta ether CFA derivative. Further assessment using both BacLight and luminescence ATP assays, confirmed that an increase in membrane permeability and reduced metabolic activity was associated with CFA treatment. Conclusions: All strains were significantly inhibited by the novel compounds studied and efficacy was related to specific structural features. Cell-membrane permeabilization was associated with CFA treatment and may account for at least a component of the mode of action of these compounds. Significance and Impact of Study: This study reports the antimicrobial action of CFA compounds against a range of Staph. aureus and MRSA strains, and provides insights into their mode of action

New Inhibitors of the FVB2 Subunit from Mammalian Kv1 Potassium Channels

The role of the redox state of KVB2 subunits in the modulation of Kv1 potassium channels has been well documented over the past few years. It has been suggested that a molecule that binds to or inhibits the aldo-keto reductase activity of KVB2 might affect the modulation of channel properties. Previous studies of possible modulators of channel activity have shown that cortisone and some related compounds are able to physically dissociate the channel components by binding to a site at the interface between � and � subunits. Herein, we describe some new inhibitors of rat brain KVB2, identified using an assay based on multiple substrate turnover. This approach allows one to focus on molecules that specifically block NADPH oxidation. These studies showed that, at 0.5 mM, 3,4-dihydroxphenylacetic acid (DOPAC) was an inhibitor of Kv�2 turnover yielding a ∼40–50% reduction in the aldehyde reductase activity of this subunit. Other significant inhibitors include the bioflavinoid, rutin and the polyphenol resveratrol; some of the known cardioprotective effects of these molecules may be attributable to Kv1 channel modulation. Cortisone or catechol caused moderate inhibition of KVB2 turnover, and the aldo-keto reductases inhibitor valproate had an even smaller effect. Despite the importance of the Kv1 channels in a number of disease states, there have been few KVB2 inhibitors reported. While the ones identified in this study are only effective at high concentrations, they could serve as tools to decipher the role of KVB2 in vivo and, eventually, inform the development of novel therapeutics

Substrate Profiling and Aldehyde Dismutase Activity of the Kvβ2 Subunit of the Mammalian Kv1 Potassium Channel

Voltage-dependent potassium channels (Kv) are involved in various cellular signalling processes by governing the membrane potential of excitable cells. The cytosolic face of these α subunit-containing channels is associated with β subunits that can modulate channel responses. Surprisingly, the β subunit of the mammalian Kv1 channels, Kvβ2, has a high level of sequence homology with the aldo-keto reductase (AKR) superfamily of proteins. Recent studies have shown that Kvβ2 can catalyze the reduction of aldehydes and, most significantly, that channel function is modulated when Kvβ2-bound NADPH is concomitantly oxidized. As a result, the redox chemistry of this subunit is crucial to understanding its role in K+ channel modulation. The present study has extended knowledge of the substrate profile of this subunit using a single turnover fluorimetric assay. Kvβ2 was found to catalyse the reduction of aromatic aldehyde substrates such as 2, 3 and 4-nitrobenzaldehydes, 4-hydroxybenzaldehyde, pyridine 2-aldehyde and benzaldehyde. The presence of an electron withdrawing group at the position para to the aldehyde in aromatic compounds facilitated reduction. Aliphatic aldehydes proved to be poor substrates. We devised a simple HPLC-based assay to identify Kvβ2 reaction products. Using this assay we showed, for the first time, that Kvβ2 can catalyze a slow aldehyde dismutation reaction using 4-nitrobenzaldehyde as substrate and have identified the products of this reaction. The ability of Kvβ2 to carry out both an aldehyde reduction and a dismutation reaction is discussed in the light of current thinking on the role of redox chemistry in channel modulation

Occupational Noise Exposure of Nightclub Bar Employees in Ireland

Due to the transposition of the EU Directive 2003/10/EC into Irish Law, the entertainment sector was obligated to comply with the requirements of the Safety, Health and Welfare at Work (General Application) Regulations 2007, Chapter 1 Part 5: Control of Noise at Work since February 2008. Compliance with the Noise Regulations was examined in 9 nightclubs in Ireland. The typical daily noise exposure of 19 bar employees was measured using 2 logging dosimeters and a Type 1 fixed position sound level meter. Physical site inspections identified nightclub noise control measures. Interviews and questionnaires were used to assess the managers and employees awareness of the noise legislation. The average bar employee daily noise exposure (LEX, 8h) was 92 dBA, almost 4 times more than the accepted legal limit. None of the venues examined were fully compliant with the requirements of the 2007 Noise Regulations, and awareness of this legislation was limited

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem statistical models, such as Design of Experiments (DoE), have been used to optimise recombinant protein production. This review explores the application of DoE in the production of recombinant proteins, focusing on prokaryotic expression systems with a specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation methods, including factorial and screening designs. It provides examples of DoE informed media optimisation and culture condition optimisation. The review concludes with a consideration of the benefits of the application of DoE in recombinant protein production

The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem, statistical models such as Design of Experiments (DoE) have been used to optimise recombinant protein production. This review examines the application of DoE in the production of recombinant proteins in prokaryotic expression systems with specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation designs. It provides examples of DoE applied to optimisation of media and culture conditions

A Short Chain NAD(H)-Dependent Alcohol Dehydrogenase (HpSCADH) from Helicobacter Pylori: a Role in Growth under Neutral and Acidic Conditions.

Toxic aldehydes produced by alcohol dehydrogenases have been implicated in the pathogenesis of Helicobacter pylori-related damage to the gastric mucosa. Despite this, the enzymes that might be responsible for producing such aldehydes have not been fully described. It was, therefore, of considerable interest to characterize the alcohol oxidizing enzymes in this pathogen. Previous work in this laboratory characterized two such H. pylori enzymes that had broad specificity for a range of aromatic alcohol substrates. An enzyme with broad specificity for aliphatic alcohols is likely to be required in order that H. pylori can metabolize the wide range of substrates encountered in the gastric mucosa. In this study we describe HpSCADH, an alcohol dehydrogenase from H. pylori 26695 with broad specificity for aliphatic alcohols. HpSCADH was classified in the cD1e subfamily of classical short chain alcohol dehydrogenases. The enzyme was a monomer of approximately 29 kDa with a preference for NAD+ as cofactor. Pyrazole was found to be a competitive inhibitor of HpSCADH. The physiological role of this enzyme was explored by construction of an HpSCADH isogenic mutant. AtpH 7.0 the mutant showed reduced growth which became more pronounced when the pH was lowered to 5.0. When pyrazole was added to wild type H. pylori cells it caused growth profiles to be reduced to those of the isogenic mutant suggesting that HpSCADH inhibition alone was responsible for growth reduction. Taken together, the data relating to the alcohol metabolizing enzymes of this pathogen indicate that they play an important role in H. pylori growth and adaptation to acidic environments. The therapeutic potential of targeting H. pylori alcohol dehydrogenases is discusse