Protein Purification Procedures Research Articles

Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use

At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation.
 We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression.
 Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis.
 The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.

Analyzing Protein Interactions by MAC-Tag Approaches.

Proteomics methods such as affinity purification (AP) and proximity-dependent labeling (PL) coupled with mass spectrometry (MS) are currently commonly utilized to define interaction landscapes. BioID is one of the PL approaches, and it employs the expression of bait proteins fused to a nonspecific biotin ligase (BirA*), to induce in vivo biotinylation of proximal proteins. We developed the multiple approaches combined (MAC)-tag workflow, which allows for both AP and BioID analysis with a single construct and with almost identical protein purification and MS identification procedures. MAC-tag is a well-established method and has been widely used. Recent developed PL tags such as BioID2 and UltraID are smaller versions of BirA* with faster labeling efficiency. We therefore incorporate these tags into our system to develop MAC2-tag (containing BioID2) and MAC3-tag (containing UltraID) to overcome potential limitations of the original MAC-tag system and broaden the spectrum of applications for MAC-tags. Here, we describe a detailed procedure for the MAC-tag system workflow including cell line generation for the MAC/MAC2/MAC3-tagged protein of interest (POI), sample preparation for AP and PL protein purification, and MS analysis.

The α-Fetoprotein Molecule has One or Two Rigid Domains Depending on the Protein Purification Procedure

Abstract: α-Fetoprotein samples isolated from human cord serum by different methods were compared using circular dichroism, scanning microcalorimetry and fluorescence spectroscopy. Their structural properties and conformational stability were appreciably discriminated. In particular, the protein molecule had either one or two relatively rigid domains which can be co-operatively disrupted by heating or urea treatment. This is due to the difference of the isolation procedures in their capabilities to strip the natural ligands from the protein molecule.

Identification, kinetics and thermodynamic analysis of novel β-galactosidase from Convolvulus arvensis seeds: An efficient agent for delactosed milk activity

The β-galactosidase was extracted and purified from 100 g of C. arvensis seeds using a variety of protein purification procedures such as ammonium sulphate fractionation, gel filtration, and finally chromatography on a cationic ion exchanger. The effects of metal ions, kinetics parameters, and glycoprotein nature were determined, as well as the optimal pH and temperature of the purified enzyme. With a high specific activity (72 units/mg), β-galactosidase was isolated to a 24-fold apparent electrophoretic homogeneity. The molecular mass of β-galactosidase was determined as monomeric, which was further confirmed by SDS-PAGE and MALDI-TOF/MS analysis, with a 45 kDa molecular weight. The enzyme has a Km of 0.33 mM and a Vmax of 42 μmol/min Lactose in milk was reduced by 38.5 and 70 % after 4 h of incubation with β-galactosidase from C. arvensis. The β-galactosidase thermal inactivation kinetic parameters ΔH°, ΔS°, and ΔG° were calculated, indicating that the enzyme undergoes significant unfolding events during denaturation. Using β-galactosidase from C. arvensis seeds, lactose hydrolysis in milk up to approx. 50 % was observed. The findings indicate the potential use of C. arvensis seeds for the production of low/delactosed milk for lactose-intolerant population.

Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility.

Recombinant E. coli producing intein-cleavable polyhydroxyalkanoate synthase fusions mediates the intracellular formation of polyhydroxyalkanoate (PHA) particles densely coated with intein-cleavable target protein fusion. These PHA particles can be efficiently purified from lysed cells. The self-cleaving intein performs as a bio-linker between the PHA synthase and the target protein. The tagless target protein can be released as pure soluble protein from the PHA particles by a simple pH reduction to 6.0. Here we describe that PHA particles serve as bioseparation resin for purification of soluble target proteins with pharmaceutical grade purity, similar to commercial affinity separation technologies. This cost-effective technique does not involve multiple complicated protein purification procedures, and we have exploited this approach to purify six target proteins: green fluorescent protein (GFP) from A. victoria, antigen Rv1626 from M. tuberculosis, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies—IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

Recombinant N-glycosylation isoforms of Legume lectins: Production and purification from Nicotiana benthamiana leaves following RuBisCO depletion

An efficient purification of recombinant proteins often requires a high ratio of recombinant to host proteins. In plants, Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant leaf protein, thus strongly impacting purification yield. Here, we describe a simple and robust purification procedure for recombinant proteins based on a differential precipitation of RuBisCO. In this context, four Legume lectin domains of Arabidopsis thaliana which belong to receptor-like kinases and cell wall proteins were produced from Nicotiana benthamiana leaves. The recombinant proteins exhibit a unique lectin domain consisting of around 250 amino acid residues with several predicted N-glycosylation sites and a six His-tag at the N-terminus. After ammonium sulphate precipitation of total soluble proteins, depletion of RuBisCO was obtained using citrate and succinate buffers during the salting-in step: this depletion was pH-dependent and the presence of di- or tri-carboxylic acids was required. The depleted protein extracts were then subjected to two chromatographic steps which were used in the negative mode to submit a protein fraction enriched as much as possible in recombinant lectin domains to a third chromatographic step (immobilized metal-ion chromatography). Three of the Legume lectin domains were purified near to homogeneity and revealed multiple N-glycosylation isoforms, particularly those from receptor-like kinases, which were characterised using specific lectins and deglycosylation enzymes. The production and purification of recombinant lectin domains will facilitate their biochemical characterisation in the context of cell-to-cell signalling and cell wall organisation.

Mass Spectrometry Analysis of Intact Proteins from Crude Samples.

Analysis of intact proteins by native mass spectrometry has emerged as a powerful tool for obtaining insight into subunit diversity, post-translational modifications, stoichiometry, structural arrangement, stability, and overall architecture. Typically, such an analysis is performed following protein purification procedures, which are time consuming, costly, and labor intensive. As this technology continues to move forward, advances in sample handling and instrumentation have enabled the investigation of intact proteins in situ and in crude samples, offering rapid analysis and improved conservation of the biological context. This emerging field, which involves various ion source platforms such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) for both spatial imaging and solution-based analysis, is expected to impact many scientific fields, including biotechnology, pharmaceuticals, and clinical sciences. In this Perspective, we discuss the information that can be retrieved by such experiments as well as the current advantages and technical challenges associated with the different sampling strategies. Furthermore, we present future directions of these MS-based methods, including current limitations and efforts that should be made to make these approaches more accessible. Considering the vast progress we have witnessed in recent years, we anticipate that the advent of further innovations enabling minimal handling of MS samples will make this field more robust, user friendly, and widespread.

Combined proximity labeling and affinity purification-mass spectrometry workflow for mapping and visualizing protein interaction networks.

Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP-MS and BioID data has been shown to comprehensively characterize a protein's molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP-MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform ( http://proteomics.fi ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP-MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.

Optimisation of urine sample preparation for shotgun proteomics

AbstractUrine reflects the renal function and urinary and kidney systems, but it may also reflect the presence of cancer in other parts of the body. Urine also has potential for providing prognostic information during therapeutic treatments thanks to non-invasive monitoring. A quick and reproducible protein purification procedure is essential to allow data comparison between proteomic studies in urine biomarker discovery. The article describes a simple, reproducible and cheap sample preparation procedure with a maximum protein yield (400 µg) obtained from only 10 mL of urine utilising cut-off filter desalting and digestion. The reported procedure removes yellowish background coloration residues and thus prevents the errors in spectrophotometric protein concentration determination. Different extraction solvents used in the presented procedure point to the possibility of partial elimination of abundant proteins (albumin and keratin family), as well as to the improvement of the sequence coverage of proteins identified, which helps to reveal changes in the urinary proteome. With this workflow, proteins can be easily obtained on standard laboratory equipment within 3 h. Data are available via ProteomeXchange with identifier PXD019738.

Yeast Surface Display System for Facilitated Production and Application of Phage Endolysin.

Bacteriophage endolysin is one of the potential alternatives of conventional antibiotics, but the intrinsic limitations of the bacterial expression system may undermine the comprehensive application of this therapeutic protein. To circumvent such limitations, we adopted a yeast surface display system as a novel expression platform for endolysin. Endolysin LysSA11 from staphylococcal phage SA11 was expressed and surface-displayed in Saccharomyces cerevisiae to exhibit sufficient antimicrobial activity against Staphylococcus aureus. Without any protein isolation or purification procedures, we showed that direct treatment of LysSA11-displaying yeast cells could accomplish a 5-log reduction of viable Staphylococcus aureus within 3 h. Furthermore, the surface-displayed LysSA11 exhibited superior stability over the soluble form of purified LysSA11 during 14 days of storage in a refrigerated environment. We suggest that the yeast surface display system is an efficient, stable, and straightforward platform for the production and antibacterial applications of endolysin.

Small-scale approach for precrystallization screening in GPCR X-ray crystallography.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor's protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda (Sf9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography.

Efficient production of gluten hydrolase Kuma030 in E. coli by hot acid treatment without chromatography

Kumamolisin from Alicyclobacillus sendaiensis strain NTAP-1 is a serine protease with collagenase activity. After molecular engineering, a kumamolisin mutant, named Kuma030, was obtained with high proteolytic activity against gluten, which might cause celiac disease. Kuma030 exhibited its potential application in industrial and medicine, while challenges remained of its large-scale purification and production. In the studies here, we successfully overexpressed the Kuma030 in E. coli BL21 (DE3) by anchoring a SUMO (Small Ubiquitin-like Modifier) fusion protein at its N-terminal end. In addition, a fast protein purification procedure was developed according to the acidophilic and thermophilic properties of Alicyclobacillus sendaiensis. After a simple acid treatment followed by a heat treatment, a total of 9.9 mg functional Kuma030 was quickly obtained form 1 L LB media culture. This purified Kuma030 was confirmed to be functional to cleave the PQ sequences in a designed protein substrate, and the gluten in actual food samples, such as whole wheat bread and beer, in a fast manner. Our studies provided an efficient strategy for the overexpression and purification of functional Kuma030 in E. coli, which might expand its broad practical applications.

Elucidating the role of an immunomodulatory protein in cancer: From protein expression to functional characterization.

Several fundamental discoveries made over the last two decades, in the field of cancer biology, have increased our understanding of the complex tumor micro- and macroenvironments. This has shifted the current empirical cancer therapies to more rationalized treatments targeting immunomodulatory proteins. From the point of identification, a protein target undergoes several interrogations, which are necessary to truly define its druggability. Here, we outline some basic steps that can be followed for in vitro characterization of a potential immunomodulatory protein target. We describe procedures for recombinant protein expression and purification including key annotations on protein cloning, expression systems, purification strategies and protein characterization using structural and biochemical approaches. For functional characterization, we provide detailed protocols for using flow-cytometric techniques in cell lines or primary cells to study protein expression profiles, proliferation, apoptosis and cell-cycle changes. This multilevel approach can provide valuable, in-depth understanding of any protein target with potential immunomodulatory effects.

Effect of C-terminal His-tag and purification routine on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of the bacteriophage T5

In this work, we studied the effect of the C-terminally attached poly-histidine tag (His-tag), as well as the peculiarities of the protein purification procedure by the immobilized metal affinity chromatography (IMAC) on the activity and structure of the metalloenzyme, l-alanyl-d-glutamate peptidase of bacteriophage T5 (EndoT5), whose zinc binding site and catalytic aspartate are located near the C-terminus. By itself, His-tag did not have a significant effect on either activity or folding of the polypeptide chain, nor on the binding of zinc and calcium ions to the protein. However, the His-tagged EndoT5 samples had low shelf-life, with storage of these samples resulting in an increased propensity for protein self-association and decreased enzymatic activity of EndoT5. Furthermore, disastrous effects on the activity of the enzyme were exerted by the presence of imidazole and nickel ions accompanying metal chelate chromatography. The activity of the protein can be restored by thorough washing off of these low molecular impurities via the prolonged dialysis of the His-tagged EndoT5 samples at the specifically elaborated conditions.

High-efficient bacterial production of human ApoA-I amyloidogenic variants.

Apolipoprotein A-I (ApoA-I)-related amyloidosis is a rare disease caused by missense mutations in the APOA1 gene. These mutations lead to protein aggregation and abnormal accumulation of ApoA-I amyloid fibrils in heart, liver, kidneys, skin, nerves, ovaries, or testes. Consequently, the carriers are at risk of single- or multi-organ failure and of need of organ transplantation. Understanding the basic molecular structure and function of ApoA-I amyloidogenic variants, as well as their biological effects, is, therefore, of great interest. However, the intrinsic low stability of this type of proteins makes their overexpression and purification difficult. To overcome this barrier, we here describe an optimized production and purification procedure for human ApoA-I amyloidogenic proteins that efficiently provides between 46 mg and 91 mg (depending on the protein variant) of pure protein per liter of Escherichia coli culture. Structural integrity of the amyloidogenic and native ApoA-I proteins were verified by circular dichroism spectroscopy and intrinsic fluorescence analysis, and preserved functionality was demonstrated by use of a lipid clearance assay as well as by reconstitution of high-density lipoprotein (HDL) particles. In conclusion, the use of the described high-yield protein production system to obtain amyloidogenic ApoA-I proteins, and their native counterpart, will enable molecular and cellular experimental studies aimed to explain the molecular basis for this rare disease.

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system

The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition the amount of purified protein is usually in the μg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. In addition, a one-step purification procedure is often not sufficient to obtain pure protein preparations. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. The first chromatographic step is the capture step followed by a desalting step. The final purification was done using size exclusion chromatography. This set-up reduces the overall-time needed for protein production, needs minimal operator invention, is easy to handle and thus increases the throughput.

Expression and characterization of recombinant rattusin, an α-defensin-related peptide with a homodimeric scaffold formed by intermolecular disulfide exchanges

Rattusin is an α-defensin-related peptide isolated from the small intestine of rats. The primary sequence of linear rattusin is composed of 31 amino acids containing five cysteines with a unique spacing pattern. It forms a homodimeric scaffold in which the primary structure occurs in an antiparallel fashion formed by five intermolecular disulfide (SS) bonds. Rattusin is a highly potent antibiotic, which not only exhibits broad-spectrum antimicrobial activity, but also maintains its antimicrobial activity at physiological salt concentrations. Therefore, to develop new antibiotics based on rattusin, structural and functional studies of rattusin should be performed. For this purpose, large amounts of linear rattusin precursor must be obtained through appropriate preparation methods. Therefore, we established a mass production technique for linear rattusin by using recombinant protein expression and purification procedures. We verified that structure and activity of the recombinant rattusin are identical to the chemically synthesized rattusin. The described method for producing recombinant rattusin provides a high yield of rattusin, which can be used to study the biochemical and functional properties of rattusin and for the development of rattusin-based peptide antibiotics.

Engineering of an Episomal Plasmid Suitable for High-Throughput Expression in Pichia pastoris.

This study describes the design and evaluation of an expression vector for Pichia pastoris (pPICZαBHF), which is based on the commercial vector construct pPICZαB. The performance of pPICZαBHF was evaluated with red fluorescent protein as a reporter. Additional His- and Flag-tags on the N-terminal ensured a simplified protein purification procedure. Transformation efficiency, expression level and plasmid maintenance were studied in order to test the functionality and usefulness of the constructed vector. We found that high transformation efficiencies were achieved using pPICZαBHF plasmid for yeast cell transformation in comparison with the commercial vector pPICZαB, which has to be integrated into the Pichia genome. However, expression levels of the recombinant protein were generally lower compared to the commercial construct. Recombinant plasmids were shown to be maintained in cells for approximately five days. Although pPICZαBHF may not be suitable for the production of high levels of recombinant protein, the simplicity of this P. pastoris expression system may still be of interest for the expression of proteins involved in cofactor regeneration or the expression of reporter genes. In addition, high transformation efficiency of pPICZαBHF may be beneficial for the applications such as high-throughput screening of mutant gene libraries.

Development of aptamers against unpurified proteins.

SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples.