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Critical Reviews in Biotechnology Jun 2020Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human... (Review)
Review
Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human diseases. Large amounts of high-quality amyloid proteins are required for better probing of their aggregation and neurotoxicity. Due to their intrinsic hydrophobicity, it is a challenge to obtain amyloid proteins with high yield and purity, and they have attracted the attention of researchers from all over the world. The rapid development of bioengineering technology provides technical support for obtaining large amounts of recombinant amyloidogenic proteins. This review discusses the available expression and purification methods for three amyloid proteins including amyloid β-protein, tau, and α-synuclein in microbial expression systems, especially , and discusses the advantages and disadvantages of these methods. Importantly, these protocols can also be referred to for the expression and purification of other hydrophobic proteins.
Topics: Amyloidogenic Proteins; Escherichia coli; Escherichia coli Proteins; Humans; Proteostasis Deficiencies; alpha-Synuclein; tau Proteins
PubMed: 32202164
DOI: 10.1080/07388551.2020.1742646 -
Biochimie Feb 2018The present review deals with the place of single chain oligonucleotide ligands (aptamers) in affinity chromatography applied to proteins. Aptamers are not the only... (Review)
Review
The present review deals with the place of single chain oligonucleotide ligands (aptamers) in affinity chromatography applied to proteins. Aptamers are not the only affinity ligands available but they represent an emerging and highly promising route that advantageously competes with antibodies in immunopurification processes. A historical background of affinity chromatography from the beginning of the discipline to the most recent outcomes is first presented. Then the focus is centered on aptamers which represent the last step so far to the long quest for affinity ligands associating very high specificity, availability and strong stability against most harsh cleaning agents required in chromatography. Then technologies of ligand selection from large libraries followed by the most appropriate chemical grafting approaches are described and supported by a number of bibliographic references. Experimental results assembled from relevant published paper are reported; they are selected by their practical applicability and potential use at large scale. The review concludes with specific remarks and future developments that are expected in the near future to turn this technology into a large acceptance for preparative applications.
Topics: Aptamers, Nucleotide; Chromatography, Affinity; Proteins
PubMed: 29054800
DOI: 10.1016/j.biochi.2017.10.008 -
Methods in Molecular Biology (Clifton,... 2018When it comes to crystallization each protein is unique. It can never be predicted beforehand in which condition the particular protein will crystallize or even if it is... (Review)
Review
When it comes to crystallization each protein is unique. It can never be predicted beforehand in which condition the particular protein will crystallize or even if it is possible to crystallize. Still, by following some simple checkpoints the chances of obtaining crystals are increased. The primary checkpoints are purity, stability, concentration, and homogeneity. High-quality protein crystals are needed. This protocol will allow an investigator to: clone, express, and crystallize a protein of interest.
Topics: Actinin; Cloning, Molecular; Crystallography, X-Ray; Gene Expression; Humans; Recombinant Proteins
PubMed: 29423850
DOI: 10.1007/978-1-4939-7546-4_9 -
Current Protocols in Protein Science Feb 2016Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the... (Review)
Review
Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.
Topics: Bacterial Proteins; Escherichia coli; Membrane Proteins; Recombinant Fusion Proteins
PubMed: 26836409
DOI: 10.1002/0471140864.ps2915s83 -
Protein Expression and Purification Feb 2021Streptococcus pneumoniae is a gram-positive bacterial pathogen causing invasive pneumonia, meningitis, otitis media, and bacteremia. Owing to the current pitfalls of...
Streptococcus pneumoniae is a gram-positive bacterial pathogen causing invasive pneumonia, meningitis, otitis media, and bacteremia. Owing to the current pitfalls of polysaccharide and polysaccharide-conjugate vaccines, protein vaccines are considered promising candidates against pneumonia. Pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) are virulence proteins showing good immunogenicity and protective effects against S. pneumoniae strains in mice. In this study, we expressed the fusion protein PsaA-PspA, which consists of PsaA and the N-terminal region of PspA family 1 and 2, in Escherichia coli. We describe a novel and effective method to purify PsaA-PspA using hydroxyapatite and two-step chromatography. After determining the optimal induction conditions and a series of purification steps, we obtained PsaA-PspA fusion protein with over 95% purity at a final yield of 22.44% from the starting cell lysate. The molecular weight of PsaA-PspA was approximately 83.6 kDa and its secondary structure was evaluated by circular dichroism. Immunization with the purified protein induced high levels of IgG antibodies in mice. Collectively, these results demonstrate that our purification method can effectively produce high-purity PsaA-PspA fusion protein with biological activity and chemical integrity, which can be widely applied to the purification of other PspA subclass proteins.
Topics: Adhesins, Bacterial; Animals; Antibodies, Bacterial; Bacterial Proteins; Escherichia coli; Female; Gene Expression; Immunoglobulin G; Mice; Mice, Inbred BALB C; Recombinant Fusion Proteins; Streptococcus pneumoniae
PubMed: 33122039
DOI: 10.1016/j.pep.2020.105782 -
Cold Spring Harbor Protocols Feb 2021Obtaining high quantities of a specific protein directly from native sources is often challenging, particularly when dealing with human proteins. To overcome this...
Obtaining high quantities of a specific protein directly from native sources is often challenging, particularly when dealing with human proteins. To overcome this obstacle, many researchers take advantage of heterologous expression systems by cloning genes into artificial vectors designed to operate within easily cultured cells, such as , (yeast), and several varieties of insect and mammalian cells. Heterologous expression systems also allow for easy modification of the protein to optimize expression, mutational analysis of specific sites within the protein and facilitate their purification with engineered affinity tags. Some degree of purification of the target protein is usually required for functional analysis. Purification to near homogeneity is essential for characterization of protein structure by X-ray crystallography or nuclear magnetic resonance (NMR) and characterization of the biochemical and biophysical properties of a protein, because contaminating proteins almost always adversely affect the results. Methods for producing and purifying proteins in several different expression platforms and using a variety of vectors are introduced here.
Topics: Cloning, Molecular; Gene Expression; Genetic Vectors; Proteins; Proteomics; Recombinant Fusion Proteins
PubMed: 33272973
DOI: 10.1101/pdb.top102129 -
Methods in Molecular Biology (Clifton,... 2020Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and... (Review)
Review
Expressed protein ligation is a simple and powerful method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size. This methodology has been developed based on the knowledge obtained from protein splicing. Protein splicing is a multistep biochemical reaction that includes the concomitant cleavage and formation of peptide bonds carried out by self-processing domains named inteins. The natural substrates of protein splicing are essential proteins found in intein-containing organisms; inteins are also functional in nonnative frameworks and can be used to alter nearly any protein's primary amino acid sequence. Accordingly, different reactivity features of inteins have been largely exploited to manipulate proteins in countless methods encompassing fields from biochemical research to the development of biotechnological applications including the study of disease progression and validation of potential drug candidates. Here, we review almost three decades of research to uncover the chemical and biochemical enigmas of protein splicing and the development of inteins as potent protein engineering tools.
Topics: Biotechnology; Isotope Labeling; Peptides, Cyclic; Protein Engineering; Protein Splicing; Recombinant Proteins
PubMed: 32144661
DOI: 10.1007/978-1-0716-0434-2_2 -
Current Protocols in Protein Science Nov 2014Purification of human IL-1β is used in this unit as an example of the preparation of a soluble protein from E. coli. Bacteria containing IL-1β are lysed, and IL-1 β... (Review)
Review
Purification of human IL-1β is used in this unit as an example of the preparation of a soluble protein from E. coli. Bacteria containing IL-1β are lysed, and IL-1 β in the resulting supernatant is purified by anion-exchange chromatography, salt precipitation, and cation-exchange chromatography, and then concentrated. Finally, the IL-1 β protein is applied to a gel-filtration column to separate it from remaining higher- and lower-molecular-weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state. In addition, the purification procedure serves as an example of how to use classical protein purifications methods, which may also be used in conjunction with the affinity-based methods now more commonly used.
Topics: Escherichia coli; Humans; Interleukin-1beta; Recombinant Proteins; Solubility
PubMed: 25367009
DOI: 10.1002/0471140864.ps0602s78 -
Protein Science : a Publication of the... Jun 2024While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial...
While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.
Topics: Histidine; Polyphosphates; Nitrilotriacetic Acid; Recombinant Proteins; Humans; Proteins
PubMed: 38747394
DOI: 10.1002/pro.5021 -
Protein Expression and Purification Dec 2021In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like... (Review)
Review
In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios.
Topics: Antibodies, Bispecific; Antibodies, Monoclonal; Bacterial Proteins; Chromatography, Affinity; Humans; Immunoglobulin G; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Lymphokines; Protein Binding; Staphylococcal Protein A
PubMed: 34537355
DOI: 10.1016/j.pep.2021.105976