Large-scale Protein Purification Research Articles

Polymeric monolithic columns based on natural wood for rapid purification of targeted protein

With multiscale hierarchical structure, wood is suitable for a range of high-value applications, especially as a chromatographic matrix. Here, we have aimed to provide a weak anion-exchange polymeric monolithic column based on natural wood with high permeability and stability for effectively separating the targeted protein. The wood-polymeric monolithic column was synthesized by in situ polymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in wood, and coupled with diethylaminoethyl hydrochloride. The wood-polymeric monolithic column can be integrated with fast-protein liquid chromatography for large-scale protein purification. According to the results, the wood-polymeric monolithic column showed high hydrophilicity, permeability and stability. Separation experiments verified that the wood-polymeric monolithic column could purify the targeted protein (spike protein of SARS-COV-2 and ovalbumin) from the mixed proteins by ion exchange, and the static adsorption capacity was 33.04 mg mL−1 and the dynamic adsorption capacity was 24.51 mg mL−1. In addition, the wood-polymerized monolithic column had good stability, and a negligible decrease in the dynamic adsorption capacity after 20 cycles. This wood-polymerized monolithic column can provide a novel, efficient, and green matrix for monolithic chromatographic columns.

Algae as a potential source of protein meat alternatives.

With the rise of plant-based meat alternatives, there is a growing need for sustainable and nutritious sources of protein. Alga is a rich protein source, and initial studies show that it can be a good component in developing protein meat alternatives. However, there are certain limitations in their use as the need for efficient and optimal technical process in large-scale protein extraction and purification, as well as overcoming certain negative effects such as potentially harmful compounds, allergenicity issues, or sensorial affections, especially in color but also in textural and flavor characteristics. This review offers a vision of the fledgling research about using alga protein in the development of meat alternatives or supplementing meat products.

Identification and characterization of E266Q mutation in Nsp15 endoribonuclease from SARS-CoV-2 Epsilon variant.

SARS-CoV-2 is responsible for the ongoing global pandemic of COVID-19. Nsp15, an endoribonuclease from SARS-CoV-2 plays active roles in immune evasion and hence emerged as a drug target for COVID-19. Here we report the identification and characterization of a high frequency mutation in Nsp15 from the SARS-CoV-2 variant, Epsilon. First detected in California, USA in July 2020, Epsilon exhibited increased transmissibility compared to other SARS-CoV-2 variants circulating at the time. We performed multiple sequence alignment of 126 genomes of epsilon and identified four non-synonymous amino acid changes in Nsp15 (V66L, A81V, D183N and E266Q). We created these four single mutants to study their impact on Nsp15 catalysis. We also created a catalytically inactive mutant (H234A) for comparative analysis. Initial protein expressions revealed, E266Q which had a mutation rate of 4.76% exhibited very low expression levels compared to wild type (WT) whereas the inactive H234A exhibited very higher expression levels. This observation was reiterated by large-scale protein purification as well. We hypothesize that being an endoribonuclease, Nsp15 might be cleaving its own mRNA during recombinant expression, thus the activity levels of E266Q, WT, and H234A directly correlate to their corresponding expression levels. We performed a preliminary activity assay which demonstrated catalytic efficiency of E266Q is equal to or even slightly higher than WT. Enzyme kinetics experiments are underway to confirm this phenomenon. We also designed a new mutation E266A to further explore the effect of the E266 residue in Nsp15 activity. Currently, we are pursuing comparative structural and functional studies on E266Q, E266A, WT, and H234A to understand how E266Q mutation renders increased catalytic activity to Nsp15 and influences Epsilon's disease transmissibility.

Artificial Water Channels—Progress Innovations and Prospects

Artificial Water Channels—Progress Innovations and Prospects

A comparison between MBP- and NT* as N-terminal fusion partner for recombinant protein production in E. coli

Advances in structural biology have been fueled in part by developing techniques for large-scale heterologous expression and purification of proteins. Nevertheless, this step is still a bottleneck in biophysical studies of many proteins. Often, fusion proteins are used to increase expression levels, solubility, or both. Here, we compare a recently reported fusion tag, NT*, with Maltose Binding Protein (MBP), a well-known fusion tag and solubility enhancer. NT* shows high expression and solubility when used as an N-terminal fusion partner for several aggregation-prone peptides. Its efficacy in enhancing the solubility of aggregation-prone globular proteins has, however, not been tested. We find here that although the overall expression levels for NT* fusions are much higher than those for the MBP fusion, MBP was far superior for enhancing the solubility of the passenger protein. Nevertheless, the effective yield after purification from the soluble fraction of both MBP-fusion and NT*-fusion was comparable, mainly due to higher expression levels in NT*-fusion and a smaller fraction of the passenger protein net weight being locked in the fusion protein. We conclude that NT* is an excellent fusion tag to improve the overall expression of globular proteins but does not increase the passenger protein's solubility compared to MBP. Proteins that are partially soluble or can be refolded in-vitro will significantly benefit from N-terminal NT* fusions. MBP, however, still remains one of the very few options for an N-terminal fusion if the solubility of the protein after expression is critical for preserving its proper fold or activity.

Tobacco System for Studying Protein Colocalization and Interactions.

Transient protein expression in a heterologous system has been very useful in many research fields. As a plant expression system, tobacco has some unique advantages including big leaves, simple infiltration and transformation, high activity in expressing transgenes, and easy sampling for microscopy. Because of these advantages, tobacco system has been extensively used for many purposes, such as large-scale expression and purification of proteins of interest, protein colocalization, protein degradation, protein-protein interaction assays including co-immunoprecipitation (CoIP), fluorescence resonance energy transfer (FRET), and bimolecular fluorescence complementation (BiFC), transcription regulation, plant-pathogen interactions, and functional verification of small RNAs. A large number of publications have used this system and generated critical results to support their conclusions. The results obtained from tobacco system are highly reproducible and mostly consistent with those generated from traditional techniques, indicating its reliability. Here we describe a protocol for studying protein-protein interactions in tobacco system, which could be applied to multiple experimental purposes as the procedure of tobacco leaf infiltration is basically shared among them.

Sample Displacement Batch Chromatography of Proteins.

In downstream processing, large-scale chromatography plays an important role. For its development, screening experiments followed by pilot-plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and upscaling of the chromatography by a factor of one hundred. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary, we provide a protocol, which should be easily adaptable for the chromatographic large-scale purification of other proteins, in the laboratory as well as in the manufacturing of biopharmaceuticals. These protocols cover the initial piloting steps for establishing a large-scale sample batch chromatography. The results from the piloting steps may also be applied for packed columns for performing simulated-moving-bed (SMB) chromatography rather than batch chromatography.

Screening and Production of Recombinant Human Proteins: Protein Production in E. coli.

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.

Screening and Production of Recombinant Human Proteins: Protein Production in Insect Cells.

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

Production of Recombinant Transmembrane Proteins from Mammalian Cells for Biochemical and Structural Analyses.

Eukaryotic integral membrane proteins are key components of various biological processes. Because they are implicated in multiple diseases, it is important to understand their mechanism of action by elucidating their structure and function. Complex technical challenges associated with the generation of recombinant membrane proteins severely impair our ability to understand them using structural and biochemical methods. Here, we provide a detailed procedure to address and mitigate difficulties involved in the large-scale heterologous overexpression and purification of eukaryotic membrane proteins using HEK293S GnTi- cells transduced with baculovirus. Two human proteins, hDHHC15 and hPORCN, are presented as examples, with step-by-step instructions for transient transfection and generation of baculoviruses, followed by overexpression and purification from HEK293S GnTi- cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Small-scale protein expression in mammalian HEK293T cells Basic Protocol 2: Generation of baculovirus from Sf9 (insect) cells Alternate Protocol: Enumeration-free method for generating P2 viral stock Support Protocol 1: Small-scale transduction of HEK293T cells with P2 baculovirus Basic Protocol 3: Large-scale viral transduction of HEK293S GnTi- cells Support Protocol 2: Large-scale membrane preparation from HEK293S GnTi- cells Basic Protocol 4: Large-scale purification of membrane proteins from HEK293S GnTi- cells.

Large-Scale Expression and Purification of Mumps Virus Hemagglutinin-Neuraminidase for Structural Analyses and Glycan-Binding Assays.

Many viruses utilize cell-surface glycans as receptors for host cell entry. Viral surface glycoproteins specifically interact with glycan motifs, which strongly contributes to viral tropism. Recently, the interactions between host cell glycan receptors and the mumps virus (MuV) hemagglutinin-neuraminidase (MuV-HN) protein were characterized by determining the co-crystal structure of MuV-HN in complex with glycan receptors. Here, we describe protocols for large-scale expression, purification and crystallization of MuV-HN proteins for structural analyses and glycan-binding assays with the overarching goal of investigating glycan-protein interactions.

Size of protein is a major factor that affects retention on preparative IMAC columns

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6 × histidine, (His)6‐tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow‐rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine‐containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow‐through (output). Data obtained suggest that regardless of the flow‐rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium‐sized proteins (~50 kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.Support or Funding InformationWelch Foundation Grant # AN‐0008 SFASU CoSM SURE 2018This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae.

This chapter outlines a protocol to assess viability for large-scale protein production and purification for selected targets from an initial medium-throughput cloning strategy. Thus, one can assess a broad number of potential candidate proteins, mutants, or expression variants using an empirically minimalistic approach. In addition, a key output from this protocol is utilization of Saccharomyces cerevisiae as a means for the efficient screening and production of purified proteins. The primary focus in this protocol is overexpression of polytopic integral membrane proteins though methods can be equally applied to soluble proteins. The protocol starts with outlining high-throughput (sans robotics) cloning of expression proteins into a dual-tag yeast expression plasmid. These membrane proteins are then screened for expression level, detergent solubilization, initial purity, and chromatography characteristics. Both small- and large-scale expression methods are discussed along with fermentation.

Size of Protein is a Major Factor that Affects Retention on Preparative IMAC Columns.

Immobilized metal affinity chromatography (IMAC) is a specific high-capacity technique used in large-scale purification of proteins. IMAC exploits the ability of immobilized metal ions to form coordination bonds with atoms in the side chains of certain amino acids. The technique is generally robust. However, several factors still affect column binding capacity, retention, yield and purity of proteins during IMAC. It was observed that the recovery of 6× histidine, (His)6-tagged proteins from metal affinity columns differ significantly depending on the size of the protein. To test this observation, we determined the effect of protein size, flow-rate, number and position of (His)6 tag on the retention of highly expressing proteins on commercial Ni2+ and Co2+ IMAC columns. All experiments were performed in phosphate buffer to eliminate interference of amine-containing buffers with the binding of the (His)6 tag to the columns. Column retention was determined as the ratio of protein of interest in the supernatant (input) to flow-through (output). Data obtained suggest that regardless of the flow-rate, (His)6 tag position and number, the size of protein is a major factor affecting column retention and therefore recovery during column IMAC purification. Small and medium-sized proteins (~ 50kDa) have higher column retention than bigger proteins, resulting in higher recovery. These outcomes provide important information to consider when performing IMAC.

Soluble T-cell receptor design influences functional yield in an E. coli chaperone-assisted expression system.

There is a quest for production of soluble protein of high quality for the study of T-cell receptors (TCRs), but expression often results in low yields of functional molecules. In this study, we used an E. coli chaperone-assisted periplasmic production system and compared expression of 4 different soluble TCR formats: single-chain TCR (scTCR), two different disulfide-linked TCR (dsTCR) formats, and chimeric Fab (cFab). A stabilized version of scTCR was also included. Additionally, we evaluated the influence of host (XL1-Blue or RosettaBlueTM) and the effect of IPTG induction on expression profiles. A celiac disease patient-derived TCR with specificity for gluten was used, and we achieved detectable expression for all formats and variants. We found that expression in RosettaBlueTM without IPTG induction resulted in the highest periplasmic yields. Moreover, after large-scale expression and protein purification, only the scTCR format was obtained in high yields. Importantly, stability engineering of the scTCR was a prerequisite for obtaining reliable biophysical characterization of the TCR-pMHC interaction. The scTCR format is readily compatible with high-throughput screening approaches that may enable both development of reagents allowing for defined peptide MHC (pMHC) characterization and discovery of potential novel therapeutic leads.

Seeding Protein Crystallization with Cross-Linked Protein Crystals

Protein crystallization is of great importance because protein crystals have a number of different important applications, including large-scale purification of proteins, determination of protein structure, nanoparticle preparation, and theoretical studies of crystallization. An approach often used to efficiently crystallize proteins is the use of nucleants or seeds (small fragments of protein crystals) that can help increase the probability of protein crystallization. Due to the very positive effect that seeding has on protein crystallization, seeds are now widely accepted and utilized in practical protein crystallization. Here, we show that cross-linked protein crystals (CLPCs), which retain the crystal structure but are much more stable than non-cross-linked crystals, can also be used as a new type of seed for promoting protein crystallization. Seeding with CLPCs has effects on both the reproducibility and screening of protein crystals and could improve the optical perfection (well-defined facets) of protein crystals and the probability of obtaining protein crystals. In addition, the cross-linked protein crystals may reduce the concentration of protein molecules needed to obtain protein crystals. Furthermore, CLPCs are very stable in air, and no protective medium is necessary for the long-term storage of CLPCs. This feature makes the CLPC seeding method a potentially powerful technique in practical protein crystallization on either a laboratorial or an industrial scale.

Transient Expression of Recombinant Membrane-eGFP Fusion Proteins in HEK293 Cells.

Membrane proteins play important roles in many biological processes and are a major drug target. However, only a limited number of structures of eukaryotic membrane proteins have been determined so far. Besides the challenges in crystallizing these proteins, one of the main bottlenecks in structure determination is their recombinant expression. The mammalian HEK293 cell line provides a natural environment for expression of eukaryotic membrane proteins but optimization of transfection and cultivation time is often necessary to yield amounts of protein suitable for structural studies.Here we describe a detailed protocol for expression and purification of membrane proteins from HEK293 cells with an example of the human peptide transporter, PepT2. In the first part, we focus on the expression optimization by changing transfection protocol and cultivation time. In the second part, we describe a robust protocol for large-scale expression and purification of membrane proteins based on affinity chromatography and gel filtration.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

Prediction of the Reverse Micellar Extraction of Papain Using Dissipative Particle Dynamics Simulation.

Reverse micellar extraction is a promising technology for large-scale protein purification, but its molecular interaction mechanisms have not been thoroughly characterized. In this study, a dissipative particle dynamics (DPD) molecular simulation method was employed to study the interactions among the surfactant, organic phase, water, and proteins on the mesoscopic scale. This study simulated the self-assembly process of the reverse micelle extraction of papain. The results showed that the papain could be extracted by a CTAB/isooctane/n-hexanol system, which was validated by extraction experiments. The optimized extraction recovery was 76.9%. This study elucidates the molecular process of the reverse micellar extraction of proteins and provides a method to predict its efficacy.