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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 Enzymology 2015X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification... (Review)
Review
X-ray crystallography remains the most robust method to determine protein structure at the atomic level. However, the bottlenecks of protein expression and purification often discourage further study. In this chapter, we address the most common problems encountered at these stages. Based on our experiences in expressing and purifying antimicrobial efflux proteins, we explain how a pure and homogenous protein sample can be successfully crystallized by the vapor diffusion method. We present our current protocols and methodologies for this technique. Case studies show step-by-step how we have overcome problems related to expression and diffraction, eventually producing high-quality membrane protein crystals for structural determinations. It is our hope that a rational approach can be made of the often anecdotal process of membrane protein crystallization.
Topics: Animals; Bacteria; Bacterial Proteins; Crystallography, X-Ray; Detergents; Diffusion; Gene Expression; Humans; Membrane Proteins; Models, Molecular; Volatilization
PubMed: 25950974
DOI: 10.1016/bs.mie.2014.12.018 -
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 -
Applied Microbiology and Biotechnology Oct 2018Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major... (Review)
Review
Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin's activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.
Topics: Animals; Chromatography, Affinity; Heparin; Humans; Proteins
PubMed: 30094590
DOI: 10.1007/s00253-018-9263-3 -
Methods (San Diego, Calif.) Oct 2015Characterization of exosomal cargo is of significant interest because this cargo can provide clues to exosome biogenesis, targeting, and cellular effects and may be a... (Review)
Review
Characterization of exosomal cargo is of significant interest because this cargo can provide clues to exosome biogenesis, targeting, and cellular effects and may be a source of biomarkers for disease diagnosis, prognosis and response to treatment. With recent improvements in proteomics technologies, both qualitative and quantitative characterization of exosomal proteins is possible. Here we provide a brief review of exosome proteomics studies and provide detailed protocols for global qualitative, global quantitative, and targeted quantitative analysis of exosomal proteins. In addition, we provide an example application of a standard global quantitative analysis followed by validation via a targeted quantitative analysis of urine exosome samples from human patients. Advantages and limitations of each method are discussed as well as future directions for exosome proteomics analysis.
Topics: Amino Acid Sequence; Annexins; Antigens, CD; Bibliometrics; Biological Transport; Biomarkers; DNA-Binding Proteins; Endosomal Sorting Complexes Required for Transport; Exosomes; Humans; Mass Spectrometry; Molecular Sequence Data; Protein Processing, Post-Translational; Proteome; Proteomics; Transcription Factors
PubMed: 25837312
DOI: 10.1016/j.ymeth.2015.03.018 -
Protein Science : a Publication of the... Mar 2016Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding... (Review)
Review
Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare.
Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Crystallization; Crystallography; Entropy; Humans; Maltose-Binding Proteins; Models, Molecular; Molecular Chaperones; Mutation; Peptides; Protein Conformation; Recombinant Fusion Proteins
PubMed: 26682969
DOI: 10.1002/pro.2863 -
Journal of Visualized Experiments : JoVE May 2021Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key...
Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.
Topics: Chromatography, Affinity; Proteins; Proteomics; Vacuum
PubMed: 34096913
DOI: 10.3791/62075 -
Protein Expression and Purification Aug 2020Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for...
Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.
Topics: Adaptor Proteins, Signal Transducing; Cloning, Molecular; Cytoskeletal Proteins; Gene Expression; Humans; Nuclear Proteins; Recombinant Fusion Proteins
PubMed: 32217127
DOI: 10.1016/j.pep.2020.105630 -
Biochimica Et Biophysica Acta.... Jan 2021G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid...
G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both G and G heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.
Topics: Detergents; Humans; Polymethacrylic Acids; Receptors, Neurotensin; Recombinant Proteins; Solubility
PubMed: 32810489
DOI: 10.1016/j.bbamem.2020.183441