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Scientific Reports Aug 2018The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may...
The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein's stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89-93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.
Topics: Chromatography, Affinity; Chromatography, High Pressure Liquid; Gene Expression; Genes, Reporter; Humans; Peptides; Pharmaceutical Preparations; Protein Engineering; Recombinant Fusion Proteins
PubMed: 30097621
DOI: 10.1038/s41598-018-30435-4 -
Bioscience Reports Jul 2021Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based...
Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.
Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cost-Benefit Analysis; DNA-Directed DNA Polymerase; Escherichia coli; Escherichia coli Proteins; Light; Mutation; Optogenetics; Protein Binding; Protein Engineering; Protein Multimerization; Recombinant Fusion Proteins; Time Factors; Workflow; Xenopus Proteins; Xenopus laevis
PubMed: 34142112
DOI: 10.1042/BSR20210800 -
Bioorganic & Medicinal Chemistry Letters Feb 2020Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high...
Solid-phase resins functionalized with poly-deoxythymidine (dT) oligos facilitate purification of poly-adenylated molecules from solution through high affinity, high selectivity base-pairing interactions. These resins are commonly used to purify messenger RNA (mRNA) from complex biological mixtures as well as mRNA-protein fusion molecules for mRNA Display selections. Historically, dT-conjugated cellulose was the primary resin for poly-dA purification, but its scarcity has prompted the development of alternative resins, most notably dT-functionalized magnetic beads. In order to develop a cost-effective alternative to commercially available poly-dT resins for large-scale purifications of mRNA-protein fusions, we investigated the purification properties of dT-conjugated Oligo Affinity Support resin (dT-OAS) alongside poly-dT magnetic beads and dT-cellulose. dT-OAS was found to have the highest dA oligo binding capacity at 4 pmol/µg, followed by dT-magnetic beads (1.1 pmol/µg) and dT-cellulose (0.7 pmol/µg). To determine the resin specificity in the context of a complex biological mixture, we translated mRNA-protein fusions consisting of a radiolabeled Her2 affibody fused to its encoding mRNA. Commercial dT-cellulose showed the highest mRNA-affibody purification specificity, followed by dT-OAS and dT-magnetic beads. Overall, dT-OAS showed exceptionally high binding capacity and low background binding, making it an attractive alternative for large-scale mRNA purification and mRNA Display library enrichment.
Topics: Cellulose; Chromatography, Affinity; Isotope Labeling; Magnetics; Poly A; RNA, Messenger; Recombinant Fusion Proteins
PubMed: 31919017
DOI: 10.1016/j.bmcl.2019.126934 -
Lab on a Chip Dec 2017Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar... (Review)
Review
Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. A number of μFFE separation modes have been demonstrated, including free zone (FZ), micellar electrokinetic chromatography (MEKC), isoelectric focusing (IEF) and isotachophoresis (ITP). Approximately 60 articles have been published since the first μFFE device was fabricated in 1994. We anticipate that recent advances in device design, detection, and fabrication, will allow μFFE to be applied to a much wider range of applications. Applications particularly well suited for μFFE analysis include continuous, real time monitoring and microscale purifications.
Topics: Cell Line; Electrophoresis; Equipment Design; Humans; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Proteins
PubMed: 29077103
DOI: 10.1039/c7lc01105a -
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 Expression and Purification Feb 2017Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal...
Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.
Topics: Chromatography, Affinity; Escherichia coli; Frontotemporal Dementia; Histidine; Humans; Recombinant Fusion Proteins; tau Proteins
PubMed: 27663563
DOI: 10.1016/j.pep.2016.09.009 -
Protein Science : a Publication of the... Aug 2017Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are...
Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.
Topics: Chromatography, Affinity; Chromatography, Gel; Computational Biology; Escherichia coli; Escherichia coli Proteins; Gene Expression; High-Throughput Screening Assays; Membrane Proteins; Peptide Library; Recombinant Proteins; Time Factors
PubMed: 28543736
DOI: 10.1002/pro.3196 -
Methods (San Diego, Calif.) Feb 2014Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of... (Review)
Review
Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of RNA-protein complexes has been identified to regulate gene expression and to be important for a range of cellular functions. In contrast to protein complexes, in vitro analyses of RNA-protein complexes are hampered by the fact that recombinant expression and purification of RNA molecules is more difficult and less well established than for proteins. Here we review the current state of technology available for in vitro experiments with RNAs. We outline the possibilities to produce and purify large amounts of homogenous RNA and to perform the required quality controls. RNA-specific problems such as degradation, 5' and 3' end heterogeneity, co-existence of different folding states, and prerequisites for reconstituting RNAs with recombinantly expressed proteins are discussed. Additionally a number of techniques for the characterization of direct and indirect RNA-protein interactions are explained.
Topics: Molecular Biology; Nucleic Acid Conformation; RNA Stability; RNA, Transfer; RNA-Binding Proteins; Ribonucleoproteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription, Genetic
PubMed: 24021718
DOI: 10.1016/j.ymeth.2013.08.034 -
Nature Communications Sep 2021In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70...
In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.
Topics: Adenosine Triphosphate; HSP70 Heat-Shock Proteins; Hydrolysis; Molecular Chaperones; Molecular Docking Simulation; Protein Domains; Protein Folding; Protein Multimerization; Recombinant Proteins; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tandem Mass Spectrometry
PubMed: 34580293
DOI: 10.1038/s41467-021-25930-8 -
Acta Crystallographica. Section F,... Sep 2011The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification...
The establishment of an efficient and reliable protein-purification pipeline is essential for the success of structural genomic projects. The SSGCID Protein Purification Group at the University of Washington (UW-PPG) has established a robust protein-purification pipeline designed to purify 400 proteins per year at a rate of eight purifications per week. The pipeline was implemented using two ÄKTAexplorer 100 s and four ÄKTAprimes to perform immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. Purifications were completed in a period of 5 d and yielded an average of 53 mg highly purified protein. This paper provides a detailed description of the methods used to purify, characterize and store SSGCID proteins. Some of the purified proteins were treated with 3C protease, which was expressed and purified by UW-PPG using a similar protocol, to cleave non-native six-histidine tags. The cleavage was successful in 94% of 214 attempts. Cleaved proteins yielded 2.9% more structures than uncleaved six-histidine-tagged proteins. This 2.9% improvement may seem small, but over the course of the project the structure output from UW-PPG is thus predicted to increase from 260 structures to 318 structures. Therefore, the outlined protocol with 3C cleavage and subtractive IMAC has been shown to be a highly efficient method for the standardized purification of recombinant proteins for structure determination via X-ray crystallography.
Topics: Communicable Diseases; Genomics; Proteins
PubMed: 21904042
DOI: 10.1107/S1744309111018367