Differential Precipitation and Solubilisation of Proteins

Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g., His-Tag, Size Exclusion, etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during overexpression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilized so as to reform functional, correctly folded proteins. Here, appropriate bioinformatics tools to aid in understanding a protein’s propensity to aggregate and solubilize are explored as a backdrop for a typical protein extraction, precipitation, and selective resolubilization procedure, based on a recombinantly expressed protein

In silico and in vitro screening for potential anticancer candidates targeting GPR120

The G-protein coupled receptor - GPR120 has recently been implicated as a novel target for colorectal cancer (CRC) and other cancer managements. In this study, a homology model of GPR120S (short isoform) was generated to identify potential anti-cancer compounds targeting the GPR120 receptor using a combined in silico docking-based virtual screening (DBVS), structure-activity relationships (SAR) and in vitro screening approach. SPECS database of synthetic chemical compounds (~350,000) was screened using the developed GPR120S model to identify molecules binding to the orthosteric binding pocket followed by an AutoDock SMINA rigid-flexible docking protocol. The best 13 hit molecules were then tested in vitro to evaluate their cytotoxic activity against SW480 - human CRC cell line expressing GPR120. The test compound 1 (3-(4-methylphenyl)-2-[(2-oxo-2-phenylethyl)sulfanyl]-5,6-dihydrospiro(benzo[h]quinazoline-5,1\u27-cyclopentane)-4(3H)-one) showed ~ 90% inhibitory effects on cell growth with micromolar affinities (IC50 = 23.21-26.69 µM). Finally, SAR analysis of compound 1 led to the identification of a more active compound from the SPECS database showing better efficacy during cell-based cytotoxicity assay -5 (IC50 = 5.89-6.715 µM), while a significant reduction in cytotoxic effects of 5 was observed in GPR120-siRNA pre-treated SW480 cells. The GPR120S homology model generated, and SAR analysis conducted by this work discovered a potential chemical scaffold, dihydrospiro(benzo[h]quinazoline-5,1\u27-cyclopentane)-4(3H)-one, which will aid future research on anti-cancer drug development for CRC management

Case-Study: An Industry-Academic Engagement Study of the Brewing and Distilling Sector in Ireland

Industry-academic (I-A) engagement models refer to the collaboration between academic institutions and industry partners to co-create and develop educational programmes that are relevant and responsive to industry needs. This study consists of research conducted as part of a 5 month Convene fellowship. It examines aspects such as industry demand, training, competency, knowledge, research projects, and modes of delivery for a proposed Postgraduate Diploma (PgDip, 60 ETS) and Master of Science (MSc, 90 ECTS) in Brewing and Distilling (B&D). This study used an I-A model to capture and collate sector specific feedback and priorities. This was achieved through 40 industry surveys and 28 stakeholder interviews, conducted between February and June 2022. Notably, this engagement model was found to effectively inform curriculum development, clarify industry skill-set expectations, and elucidate sector demands, challenges, knowledge gaps, and opportunities

Structure based prediction of a novel GPR120 antagonist based on pharmacophore screening and molecular dynamics simulations

The G-protein coupled receptor, GPR120, has ubiquitous expression and multifaceted roles in modulating metabolic and anti-inflammatory processes. Recent implications of its role in cancer progression have presented GPR120 as an attractive oncogenic drug target. GPR120 gene knockdown in breast cancer studies revealed a role of GPR120-induced chemoresistance in epirubicin and cisplatin-induced DNA damage in tumour cells. Higher expression and activation levels of GPR120 is also reported to promote tumour angiogenesis and cell migration in colorectal cancer. Some agonists targeting GPR120 have been reported, such as TUG891 and Compound39, but to date development of small-molecule inhibitors of GPR120 is limited. Herein, following homology modelling of the receptor a pharmacophore hypothesis was derived from 300 ns all-atomic molecular dynamics (MD) simulations on apo, TUG891-bound and Compound39-bound GPR120S (short isoform) receptor models embedded in a water solvated lipid bilayer system. We performed comparative MD analysis on protein–ligand interactions between the two agonist and apo simulations on the stability of the “ionic lock” – a Class A GPCRs characteristic of receptor activation and inactivation. The detailed analysis predicted that ligand interactions with W277 and N313 are critical to conserve the “ionic-lock” conformation (R136 of Helix 3) and prevent GPR120S receptor activation. The results led to generation of a W277 and N313 focused pharmacophore hypothesis and the screening of the ZINC15 database using ZINCPharmer through the structure-based pharmacophore. 100 ns all-atomic molecular dynamics (MD) simulations were performed on 9 small molecules identified and Cpd 9, (2-hydroxy-N-{4-[(6-hydroxy-2-methylpyrimidin-4-yl) amino] phenyl} benzamide) was predicted to be a small-molecule GPR120S antagonist. The conformational results from the collective all-atomic MD analysis provided structural information for further identification and optimisation of novel druggable inhibitors of GPR120S using this rational design approach, which could have future potential for anti-cancer drug development studies

Approaches to Avoid Proteolysis During Protein Expression and Purification

All cells contain proteases, which hydrolyze the peptide bonds between amino acids of 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: first, inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographic 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. In this chapter, commonly used approaches to reducing or avoiding proteolysis during protein expression and purification are reviewed

Protein Extraction and Purification by Differential Solubilization

The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this process. Selectively altering the solubility of a protein is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of a polypeptide. Precipitation of proteins, released from cells upon lysis, is often used to concentrate a protein of interest before further purification steps (e.g., ion exchange chromatography, size exclusion chromatography etc). Recombinant proteins may be expressed in host cells as insoluble inclusion bodies due to various influences during overexpression. Such inclusion bodies can often be solubilized to be reconstituted as functional, correctly folded proteins. In this chapter, we examine strategies for extraction/precipitation/solubilization of proteins for protein purification. We also present bioinformatic tools to aid in understanding a protein’s propensity to aggregate/solubilize that will be a useful starting point for the development of protein extraction, precipitation, and selective re-solubilization procedures

Enzymatic in-situ transesterification of neutral lipids from simulated wastewater cultured Chlorella emersonii and Pseudokirchneriella subcapitata to sustainably produce fatty acid methyl esters

Alternative, more sustainable and environmentally positive, sources of energy are one of the current global challenges. One approach to achieving more sustainable sources of energy is to use waste from one system as a raw material for energy production, following the circular biosystem philosophy. This study successfully adopted this approach whereby microalgae strains Chlorella emersonii and Pseudokirchneriella subcapitata were bioprospected and metabolically engineered in simulated wastewater supplemented with glucose to produce neutral lipids. Using a two-step cultivation approach neutral lipid content was enhanced in Chlorella emersonii and Pseudokirchneriella subcapitata biomass. Via in-situ transesterification, these neutral lipids were subsequently bioconverted to biodiesel feedstock fatty acid methyl esters using novel solvent stable lipase(s) from Pseudomonas reinekei and Pseudomonas brenneri. The culturing of appropriate microalgae on wastewater, and bioconversion via organo-stable lipases may provide a commercially viable and sustainable biodiesel feedstock to help address the current global energy challenge

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

Isolation, Purification and Characterization of a Novel Solvent Stable Lipase From Pseudomonas Reinekei

The Pseudomonas sp. have been long recognized for their exogenous lipolytic activities yet the genus still contains a lot of unexplored strains. Due to the versatile metabolic machinery and their potential for adaptation to fluctuating environmental conditions Pseudomonas sp. are of great interest for biotechnological applications. In this study, a new extracellularly produced lipolytic enzyme from Pseudomonas sp. (P. reinekei) was purified and characterized. The production of lipase from P. reinekei (H1) was enhanced 10-fold by optimizing the nitrogen source. The 50 kDa H1 lipase was purified using negative and positive mode anion exchange chromatography. The purified lipase was active over a broad pH range (5.0-9.0) and was stable for 24h at 40°C. The lipase showed significant stability, and indeed activation, in the presence of organic solvents with log P≥ 2.0. These features render this lipase of interest as a biocatalyst for applications such as biodiesel production, detergent formulations and biodegradation of oil in the environment

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