Purification Of Recombinant Proteins Research Articles

Immunosensor for realtime monitoring of the expression of recombinant proteins during bioprocess

Recombinant protein expression and purification are crucial in modern life sciences research. A fluorescent immunosensor termed Quenchbody (Q-body) was developed for real-time monitoring of FLAG-fused protein expression. Detection results showed that the limit of detection of the 3 × FLAG peptide detected by the TAMRA-labeled anti-FLAG Q-body was as low as 3.1 nM, with a half-maximal effective concentration of 21.4 nM. Furthermore, the anti-FLAG Q-body was used for detecting different proteins fused with a FLAG-tag at the N- or C-terminal. Subsequently, the constructed Q-body was used to monitor the real-time fermentation process of single-strand DNA-binding protein in Escherichia coli. Unlike previously reported Q-bodies that widely used Fab or scFv, the present study used a full-length anti-FLAG IgG for the first time. Owing to its excellent detection speed and sensitivity, the FLAG Q-body immunosensor has the potential to quantify and monitor target recombinant proteins in multiple biological processes in real-time.

Immunization of mice with chimeric protein-loaded aluminum hydroxide and selenium nanoparticles induces reduction of Brucella melitensis infection in mice.

Due to the many problems with commercially available vaccines, the production of effective vaccines against brucellosis is a necessity. The aim of this study was to evaluate the immune responses caused by the chimeric protein consisting of trigger factor, Bp26, and Omp31 (TBO) along with aluminum hydroxide (AH/TBO) and selenium (Se/TBO) nanoparticles (NPs) as adjuvants in mouse model. Recombinant antigen expression was induced in Escherichia coli BL21 (DE3) bacteria using IPTG (isopropyl-d-1-thiogalactopyranoside). Purification and characterization of recombinant protein was conducted through NiFe3O4 NPs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. NP characteristics, including morphology and particle size, were measured in vitro. The recombinant TBO was loaded on to AH and Se NPs and were administered subcutaneously. After mice immunization, measurement of antibody titter and protection assay was performed. The average sizes of AH and Se NPs were about 60 nm and 150 nm, respectively. The enzyme-linked immunosorbent assay results showed that the serum of mice immunized by subcutaneous injection with both nanovaccines produced significant immunoglobulin G (IgG) responses against the chimeric antigen. The results of TBO-specific IgG isotype (IgG2a/IgG1) analysis showed that both AH and Se NPs induced a type to T-helper immune response. In addition, the results of the challenge with the pathogenic strain of Brucella melitensis 16M showed that vaccinated mice with AH/TBO NPs indicated a higher reduction of bacterial culture than immunized mice with Se/TBO NPs and TBO alone. The results showed that AH NPs carrying chimeric antigen can be a promising vaccine candidate against brucellosis by producing protective immunity.

Tagging Recombinant Proteins to Enhance Solubility and Aid Purification.

Protein fusion technology has had a major impact on the efficient production and purification of individual recombinant proteins. The use of genetically engineered affinity and solubility-enhancing polypeptide "tags" has a long history, and there is a considerable repertoire of these that can be used to address issues related to the expression, stability, solubility, folding, and purification of their fusion partner. In the case of large-scale proteomic studies, the development of purification procedures tailored to individual proteins is not practicable, and affinity tags have become indispensable tools for structural and functional proteomic initiatives that involve the expression of many proteins in parallel. In this chapter, the rationale and applications of a range of established and more recently developed solubility-enhancing and affinity tags is described.

Preparation and Utilization of a Versatile GFP-Protein Trap-Like System for Protein Complex Immunoprecipitation in Plants.

Protein complex immunoprecipitation (co-IP) is an in vitro technique used to study protein-protein interaction between two or more proteins. This method relies on affinity purification of recombinant epitope-tagged proteins followed by western blotting detection using tag-specific antibodies for the confirmation of positive interaction. The traditional co-IP method relies on the use of porous beaded support with immobilized antibodies to precipitate protein complexes. However, this method is time-consuming, labor-intensive, and provides lower reproducibility and yield of protein complexes. Here, we describe the implementation of magnetic beads and high-affinity anti-green fluorescent protein (GFP) antibodies to develop an in vitro GFP-protein trap-like system. This highly reproducible system utilizes a combination of small sample size, versatile lysis buffer, and lower amounts of magnetic beads to obtain protein complexes and aggregates that are compatible with functional assays, Western blotting, and mass spectrometry. In addition to protein-protein interactions, this versatile method can be employed to study protein-nucleic acid interactions. This protocol also highlights troubleshooting and includes recommendations to optimize its application.

Expression and Purification of Recombinant Multi-epitope Protein of Rhipicephalus microplus Tick and its Antigenicity in Rabbits

Expression and Purification of Recombinant Multi-epitope Protein of Rhipicephalus microplus Tick and its Antigenicity in Rabbits

Recombinant protein production for structural and kinetic studies: A case study using M. tuberculosis α-methylacyl-CoA racemase (MCR).

Modern drug discovery is a target-driven approach in which a particular protein such as an enzyme is implicated in the disease process. Commonly, small-molecule drugs are identified using screening, rational design, and structural biology approaches. Drug screening, testing and optimization is typically conducted in vitro, and copious amounts of protein are required. The advent of recombinant DNA technologies has resulted in a rise in proteins purified by affinity techniques, typically by incorporating an "affinity tag" at the N- or C-terminus. Use of these tagged proteins and affinity techniques comes with a host of issues. This chapter describes the production of an untagged enzyme, α-methylacyl-CoA racemase (MCR) from Mycobacterium tuberculosis, using a recombinant E. coli system. Purification of the enzyme on a 100mg scale using tandem anion-exchange chromatographies (DEAE-sepharose and RESOURCE-Q columns), and size-exclusion chromatographies is described. A modified protocol allowing the purification of cationic proteins is also described, based on tandem cation-exchange chromatographies (using CM-sepharose and RESOURCE-S columns) and size-exclusion chromatographies. The resulting MCR protein is suitable for biochemical and structural biology applications. The described protocols have wide applicability to the purification of other recombinant proteins and enzymes without using affinity chromatography.

Crystalline Biohybrid Materials Based on Protein Cages.

Highly ordered superstructures of nanomaterials can be synthesized using protein cages as templates for the assembly of inorganic nanoparticles. Here, we describe in detail the creation of these biohybrid materials. The approach involves computational redesign of ferritin cages, followed by recombinant protein production and purification of the new variants. Metal oxide nanoparticles are synthesized inside the surface-charged variants. The composites are assembled using protein crystallization to yield highly ordered superlattices, which are characterized, for example, with small angle X-ray scattering. This protocol provides a detailed and comprehensive account on our newly established strategy for the synthesis of crystalline biohybrid materials.

Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA

BackgroundMycoplasma gallisepticum is the primary agent of chronic respiratory disease in chickens creating important economic losses in poultry industry. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. The objective of the present study was to design, express, and purify the recombinant chimeric PvpA-pMGA protein from M.gallisepticum for using in serological diagnostic test. MethodsAntigenic regions of PvpA and pMGA proteins were predicted for designing chimeric pvpA-pMGA gene construct. The codon optimized sequence was cloned into the expression vector pET32a+ and transformed into the Escherichia coli strain BL21 (DE3). The pET32a-PvpA-pMGA recombinant plasmid was expressed and confirmed by SDS-PAGE and immunoblotting. PvpA-pMGA recombinant protein (20μg and 50μg), ts-11 vaccine strain, and S6 strain that formulated by montanide adjuvant and two control groups (PBS and adjuvant) were injected subcutaneously to six groups of chickens. ResultsHigh yield of protein was purified amount 138 mg/L by affinity batch formation method. Indirect ELISA showed the levels of antibodies in rPvpA-pMGA was significantly higher than ts-11 and S6 groups (p<0.05). The results indicated that antigen-specific response was successfully elicited by the rpMGA-PvpA in chickens. The result of the ELISA with sera collected from ts-11 and S6 groups showed that indirect PvpA-pMGA-ELISA is appropriate candidate for detection of specific antibodies against M. gallisepticum with 100% sensitivity and specificity. ConclusionsThe rPvpA-pMGA is a highly candidate immunogenic protein which induced high amount of humoral immune response. Novel rPvpA-pMGA protein could be useful for evaluation of antibody level in vaccinated poultry flocks. Highlights•This is first study to present chimeric protein rPvpA-pMGA as a candidate antigen in ELISA.•High-yield 138 mg/L purification of recombinant protein.•rPvpA-pMGA is a reliable immunogenic protein which induced high amount of humoral immune response.•rPvpA-pMGA showed high performance for evaluation of antibody level in vaccinated poultry flocks.

Development of functional anti-Gn nanobodies specific for SFTSV based on next-generation sequencing and proteomics.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, we demonstrated that SFTSV surface glycoprotein (Glycoprotein N, Gn) was a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies played a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages, including small molecular weight, high affinity, low immunogenicity, convenient production by gene engineering, etc. In this study, we combined next-generation sequencing (NGS) with proteomics technology based on affinity purification-mass spectrometry (AP-MS) and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, of which six sequences were selected for recombinant protein expression and purification. Among these six anti-Gn nanobodies, nanobody 57,493 was validated to be highly specific for Gn. The innovative high-throughput technical route developed in this study could also be expanded to the production of nanobodies specific for other viruses like SARS-CoV-2.

&lt;em&gt;In Vitro&lt;/em&gt; Cleavage Assays using Purified Recombinant &lt;em&gt;Drosophila&lt;/em&gt; Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening.

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

Generation of a recombinant version of a biologically active cell-permeant human HAND2 transcription factor from E. coli

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. It is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli strain, BL21(DE3). Next, the effect (in terms of expression) of tagging fusion tags with this recombinant protein at two different terminals was also investigated. Using affinity chromatography, we established the one-step homogeneous purification of recombinant human HAND2 fusion protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. We then showed that this purified human protein could transduce the human cells and translocate to its nucleus. The generated recombinant HAND2 fusion protein showed angiogenic potential in the ex vivo chicken embryo model. Following transduction in MEF2C overexpressing cardiomyoblast cells, this purified recombinant protein synergistically activated the α-MHC promoter and induced GFP expression in the α-MHC-eGFP reporter assay. Prospectively, the purified bioactive recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

Preparation of nickel-chelated iminodiacetate-functionalized macroporous agarose monolith using modular and clickable building blocks for affinity separation of histidine-tagged recombinant proteins

Selective separation and purification of protein from complex medium is required to completely investigate the structure and function of the target protein. In this study, a composite macroporous agarose monolith containing iminodiacetate-chelated Ni2+ ligands was synthesized for selective separation and purification of histidine-tagged recombinant proteins. The large and interconnected pores in the monolith enabled fast binding of proteins with high matrix tolerance in treating complex mediums. To realize the selective protein binding, the iminodiacetate was directly conjugated to epoxy-functionalized agarose monolith via simple chemical reactions between epoxy and imino groups. After chelated Ni2+, the composite monolith could bind histidine-tagged recombinant proteins through the coordination interaction between transition metal ions and the imidazole ring of histidine. To further increase the binding capacities of the monolith, a hydrophilic intermediate polymer chain containing multiple iminodiacetate immobilization sites was conjugated to the azide-functionalized agarose monolith via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The morphology and chemical composition of the composite agarose monolith were characterized systematically. The protein binding capacities of the obtained composite agarose monolith were subsequently investigated. The binding capacities of the composite agarose monolith towards the model proteins Gp10 and Lys84 were 0.93 and 0.51 mg/mL, respectively. The protein binding of the composite agarose monolith could be manipulated by adjusting the temperature and concentrations of imidazole. These results demonstrate that the composite agarose monolith could be used as an affinity medium for rapid separation and purification of histidine-tagged recombinant proteins from biological samples.

High-efficiency recombinant protein purification using mCherry and YFP nanobody affinity matrices.

Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post-translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real-time monitoring of protein expression, and also provides an affinity handle for one-step protein purification using a suitable affinity reagent. Here, we describe a panel of anti-GFP and anti-mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X-ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody-sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry-tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry-tag and GFP/YFP-tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real-time monitoring of protein expression levels and a high-efficiency purification step is needed.

Scalable purification of recombinant structural proteins, hagfish intermediate filament α and ɣ, from inclusion bodies for fiber formation

The purpose of this study was to determine a method to purify recombinant hagfish intermediate filament proteins, alpha and gamma, in a scalable manner. The study succeeded by having an increase in protein recovery of up to 35% when comparing centrifuge purification and the developed tangential flow purification. The proteins were approximately the same purity of 70% pure but further purification increased the purity of the proteins by 16%, based on ImageJ analysis. The developed tangential flow filtration purification and final purification methods could be easily scaled up to meet industry scale purification needs. The scaled-up processes described in this study did not interfere with fiber production or formation, indicating the methods can produce usable proteins for material development.

CASPON platform technology: Ultrafast circularly permuted caspase-2 cleaves tagged fusion proteins before all 20 natural amino acids at the N-terminus

Fusion protein technologies improve the expression and purification of recombinant proteins, but the removal of the tags involved requires specific proteases. The circularly permuted caspase-2 (cpCasp2) with its specific cleavage site, efficiently generates the untagged protein. While cleavage with cpCasp2 is possible before all 20 proteinogenic amino acids, cleavage before valine, leucine, isoleucine, aspartate and glutamate suffers from slow, and before proline extremely slow, turnover. To make the platform fusion protein process even more general such that any protein with an authentic N-terminus can be produced with high efficiency, the bacterial selection system PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection) was used to evolve cpCasp2 into a variant with a catalytic turnover two orders of magnitude higher and the ability to cleave before any amino acid. The high specificity and the stability of the original circularly permuted protease was fully retained in this mutant, while the high manufacturability was mostly retained, albeit with decreased soluble titer. Four point-mutations are responsible for this change in activity, two of which are located in or near the binding pocket of the active site. This variant was named CASPON enzyme and is a major component of the CASPase-based fusiON (CASPON) platform technology. Applicability for the production of recombinant proteins was demonstrated by enzymatic removal of the CASPON tag from five model proteins. The CASPON tag enables high soluble expressions, affinity purification and good accessibility for cleavage. The five industry-relevant proteins of interest were FGF2, TNF, GH, GCSF and PTH.

Optimized Purification of CIDRα-PfEMP1 Plasmodium falciparum Recombinant Protein with Affinity Chromatography

Interaction of Cysteine-rich Interdomain Region (CIDR)α-Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) and Endothelial Cell Receptors especially CD36 on host cells is main malaria pathogenesis, makes this domain as a malaria vaccine candidate. Recently, the development of the malaria vaccine is conducted by recombinant technology, and the purification of the CIDRα-PfEMP1 recombinant protein is a pivotal step. This study aimed to determine an optimal condition to purify the CIDRα-PfEMP1 recombinant protein by affinity chromatography through imidazole and NaCl concentration. The purified recombinant protein was visualized using SDS-PAGE and its concentration was measured using Image J software and Bradford Assay. The data were analyzed using SPSS 26 software, and the Paired T-Test analysis was conducted to compare the concentration of purified recombinant protein from two different methods. The result showed that thetarget band of purified recombinant protein was 27 kDa. The thickest target protein band was observed in purified recombinant protein using 140 mM imidazole and 300 mM NaCl. The recombinant protein concentration using Image J software was 0.025 µg/µL, while the Bradford Assay was 0.56 µg/µL. The Paired T-Test analysis has a significance value of 0.010 (p&lt;0.05), meaning there was a significant difference between the concentration measurement using Image J software and Bradford Assay. In conclusion, the optimized condition to purify the CIDRα-PfEMP1 recombinant protein by affinity chromatography was using 140 mM imidazole and 300 mM NaCl. It is suggested to measure the purified CIDRα-PfEMP1 recombinant protein concentration using the Bradford Assay method due to its convenience and sensitivity.

An engineered peptide tag-specific nanobody for immunoaffinity chromatography application enabling efficient product recovery at mild conditions

Camelid-derived nanobody is emerging as a resourceful platform for developing immunoaffinity ligands for chromatography applications. Featured by high affinity and selectivity, BC2 nanobody (BC2-Nb), which can recognize a specific epitope tag (PDRKAAVSHWQQ, termed BC2T), is potential to be developed as a general tool for recombinant protein purification. However, excessively high affinity between binding partners makes the desorption of products less efficient and limits its application. Aiming to improve elution efficiency, structure-guided mutations of BC2-Nb were conducted to adjust the structural flexibility of its antigen-binding site. Six ligand variants were obtained with their binding affinity decreasing by about 100-fold. Among them, one mutated BC2-Nb named 44D was chosen to prepare immunoaffinity resin, and its adsorption and elution performance were well characterized. The site-directed mutation led to the equilibrium dissociation constant (KD) of BC2-Nb changing from 1.4 × 10−9 M to 1.4 × 10−7 M (44D). The resin using 44D as ligand retained a static binding capacity of 19.14 mg/mL toward BC2T-fused enhanced green fluorescent protein (eGFP-BC2T). Significantly improved elution efficiency was obtained with the mutated ligand. Protein recovery reached 94% at pH 3.5 for 44D-based resin, while the resin based on original BC2-Nb could only achieve its highest recovery of 84% at pH 2. In addition, a neutral elution condition (1 M arginine containing 50% propylene glycol, pH 7.4) was also found effective, which allowed a product recovery of 95%. The resin enabled direct capturing of eGFP-BC2T from bacterial lysates, and the one-step purification with the both elution conditions could achieve a product purity of more than 90%. This study provided a promising affinity ligand, and also proved the feasibility of controlling the elution process of nanobody-based affinity resin through the strategy of binding sites modification.

Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) showing anti-S. aureus and anti-biofilm properties by elevating activities of serine protease (SspA) and cysteine protease staphopain B (SspB).

Staphylococcus aureus biofilms are the pathogenic factor in the spread of infection and are more pronounced in multidrug-resistant strains of S. aureus, where high expression of proteases is observed. Among various proteases, Serine protease (SspA) and cysteine protease Staphopain B (SspB) are known to play a key role in the biofilm formation and removal of biofilms. In earlier studies, we have reported Dibenzyl (benzo [d] thiazol-2-yl (hydroxy) methyl) phosphonate (DBTMP) exhibits anti-S. aureus and anti-biofilm properties by elevating the expression of the protease. In this study, the effect of DBTMP on the activities of SspA, and SspB of S. aureus was evaluated. The SspA and SspB genes of S. aureus ATCC12600 were sequenced (Genbank accession numbers: MZ456982 and MW574006). In S. aureus active SspA is formed by proteolytic cleavage of immature SspA, to get this mature SspA (mSspA), we have PCR amplified the mSspA sequence from the SspA gene. The mSspA and SspB genes were cloned, expressed, and characterized. The pure recombinant proteins rSspB and rmSspA exhibited a single band in SDS-PAGE with a molecular weight of 40 and 30KD, respectively. The activities of rmSspA and rSspB are 32.33 and 35.45Units/mL correspondingly. DBTMP elevated the activities of rmSspA and rSspB by docking with respective enzymes. This compound disrupted the biofilms formed by the multidrug-resistant strains of S. aureus and further prevented biofilm formation. These findings explain that DBTMP possesses anti-S. aureus and anti-biofilm features.