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Scientific Reports Oct 2017Recently, a magnetic protein was discovered, and a multimeric magnetosensing complex was validated, which may form the basis of magnetoreception. In this study, the...
Recently, a magnetic protein was discovered, and a multimeric magnetosensing complex was validated, which may form the basis of magnetoreception. In this study, the magnetic protein was firstly used in biotechnology application, and a novel convenient one-step purification and immobilization method was established. A universal vector and three linker patterns were developed for fusion expression of magnetic protein and target protein. The magnetic protein was absorbed by iron beads, followed by target protein aggregation, purification, and immobilization. GFP, employed as a reporter protein, was successfully purified from cell lysate. Subsequently, three enzymes (lipase, α-L-arabinofuranosidase, pullulanase) with different molecular sizes testified the versatility of this magnetic-based approach. The specific activities of the purified enzymes were distinctly higher than those of the traditionally purified enzymes using affinity chromatography. The lipase immobilized on iron beads presented improved thermostability and enhanced pH tolerance compared to the free enzyme. The immobilized lipase could be easily recovered and reused for maximum utilization. After 20 cycles of reutilization, the magnetically immobilized lipase retained 71% of its initial activity. This investigation may help introduce magnetic protein into biotechnology applications, and the one-step purification and immobilization method may serve to illustrate an economically viable process for industry.
Topics: Biotechnology; Cloning, Molecular; Enzymes, Immobilized; Gene Expression; Lipase; Magnetic Phenomena; Proteins
PubMed: 29042612
DOI: 10.1038/s41598-017-13648-x -
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 -
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 -
Journal of Chromatography. A Aug 2021PEGylated proteins comprise a class of value-added biopharmaceuticals. High-resolution separation techniques are required for the purification of these molecules. In...
PEGylated proteins comprise a class of value-added biopharmaceuticals. High-resolution separation techniques are required for the purification of these molecules. In this study, we discuss the application of a newly developed z laterally-fed membrane chromatography (or zLFMC) device for carrying out high-resolution purification of a PEGylated protein drug. The device used in the current study contained a stack of anion exchange (Q) membranes. The membrane bed-height of this zLFMC device being small, it could be operated at very high flow rates, at relatively low back pressures. The primary goal was to speedily and efficiently separate a mono-PEGylated protein from impurities present in the PEGylation reaction mixture. A resin-based anion exchange column having the same ligand and bed-volume was used as the control device. The purification performance of the zLFMC device and the control column were compared terms of resolution, recovery and purity. The zLFMC device outperformed the control column in terms of every metric compared in this study. Higher purity (85.4% as opposed to 77.9%) and higher recovery (28% greater) of the target mono-PEGylated protein were obtained using the zLFMC device at 20-time higher speed. These results clearly demonstrate that the zLFMC device could be a faster and more efficient alternative to resin-based columns for purification of biopharmaceuticals.
Topics: Biological Products; Chemistry, Pharmaceutical; Chromatography; Polyethylene Glycols; Proteins
PubMed: 34256267
DOI: 10.1016/j.chroma.2021.462375 -
Protein Expression and Purification Nov 2018TMEM8B-a protein is the longer, predominant isoform of the TMEM8B gene product, which is a tumor metastasis suppressor in nasopharyngeal carcinoma (NPC) and lung cancer....
TMEM8B-a protein is the longer, predominant isoform of the TMEM8B gene product, which is a tumor metastasis suppressor in nasopharyngeal carcinoma (NPC) and lung cancer. TMEM8B-a is rapidly degraded via the proteasome pathway mediated by ezrin in many NPC and lung cancer cell lines, but TMEM8B-a is not ubiquitinated. In this study, we report the recombinant production of full-length modified TMEM8B-a in mammalian cells. We used the PiggyBac transposon system to efficiently generate normal and lung cancer cell lines with stable TMEM8B-a protein expression. 293FT cells were the best host cell line to express TMEM8B-a protein. Then, we treated the stable 293FT cell lines with various small-molecule inhibitors and demonstrated that treatment with MG-132 and bortezomib, which target the proteasome and disrupt its function, could prevent TMEM8B-a degradation and induce protein expression in 293FT cells. Finally, we utilized the combination of Twin-Strep-tag and Strep-Tactin XT resin to successfully purify the TMEM8B-a protein. The final yield was estimated to be approximately 10-20 μg of the purified TMEM8B-a per 3.0 × 10 293FT cells.
Topics: Bortezomib; Cell Line; Humans; Leupeptins; Membrane Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Isoforms; Proteolysis; Recombinant Proteins; Transfection; Tumor Suppressor Proteins
PubMed: 29886078
DOI: 10.1016/j.pep.2018.06.002 -
Protein Expression and Purification Feb 2018Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides...
Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.
Topics: Cell Membrane; Chromatography, Affinity; Chromatography, Gel; Cloning, Molecular; Detergents; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Gene Expression; Humans; Plasmids; Recombinant Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 28918196
DOI: 10.1016/j.pep.2017.09.002 -
Methods in Molecular Biology (Clifton,... 2018Protein misfolding, aggregation, and amyloid formation is involved in a large number of diseases. Recombinantly expressed proteins to study the amyloid fibril formation...
Protein misfolding, aggregation, and amyloid formation is involved in a large number of diseases. Recombinantly expressed proteins to study the amyloid fibril formation process are important for mechanistic studies. We here report protocols for production, purification, and fibrillation of three different proteins commonly found in cerebral amyloid; Aβ and Tau found in Alzheimer's disease, Chronic traumatic brain injury, Corticobasal degeneration, and Progressive Supranuclear Palsy and human prion protein found in Creutzfeldt-Jakob's disease. The three protocols have in common that the protein is in a pH-neutral phosphate saline buffer during fibrillation to mimic their endogenous near physiological environment.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Chromatography, Ion Exchange; Creutzfeldt-Jakob Syndrome; Humans; Neurodegenerative Diseases; Prion Proteins; Protein Folding; Recombinant Proteins; tau Proteins
PubMed: 29886532
DOI: 10.1007/978-1-4939-7816-8_10 -
Analytical Chemistry Jan 2018
Review
Topics: Chromatography, Liquid; Protein Conformation; Proteins; Proteomics
PubMed: 29207237
DOI: 10.1021/acs.analchem.7b05007 -
Bioprocess and Biosystems Engineering Jul 2015Since about 170 years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or... (Review)
Review
Since about 170 years, salts were used to create supersaturated solutions and crystallize proteins. The dehydrating effect of salts as well as their kosmotropic or chaotropic character was revealed. Even the suitability of organic solvents for crystallization was already recognized. Interestingly, what was performed during the early times is still practiced today. A lot of effort was put into understanding the underlying physico-chemical interaction mechanisms leading to protein crystallization. However, it was understood that already the solvation of proteins is a highly complex process not to mention the intricate interrelation of electrostatic and hydrophobic interactions taking place. Although many basic questions are still unanswered, preparative protein crystallization was attempted as illustrated in the presented case studies. Due to the highly variable nature of crystallization, individual design of the crystallization process is needed in every single case. It was shown that preparative crystallization from impure protein solutions as a capture step is possible after applying adequate pre-treatment procedures like precipitation or extraction. Protein crystallization can replace one or more chromatography steps. It was further shown that crystallization can serve as an attractive alternative means for formulation of therapeutic proteins. Crystalline proteins can offer enhanced purity and enable highly concentrated doses of the active ingredient. Easy scalability of the proposed protein crystallization processes was shown using the maximum local energy dissipation as a suitable scale-up criterion. Molecular modeling and target-oriented protein engineering may allow protein crystallization to become part of a platform purification process in the near future.
Topics: Crystallization; Molecular Weight; Organic Chemicals; Proteins; Salts; Solubility; Temperature
PubMed: 25700885
DOI: 10.1007/s00449-015-1374-y -
Biotechnology Letters Feb 2015Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The ever-increasing knowledge about the... (Review)
Review
Immunoglobulin A (IgA) is the most abundant antibody class in the human body and has a unique role in mediating immunity. The ever-increasing knowledge about the potential of IgAs has renewed interest in this antibody class for therapeutic use against a variety of infectious and malignant diseases, and as a preventive agent for mucosal pathogens. Despite the considerable therapeutic potential of IgA the exploration thereof has often been hampered due to difficulties in producing and purifying desired quantities. Large amounts of pure IgA will be required for in vivo studies. This work reviews current achievements and bottlenecks in upstream and downstream processing of recombinant IgA from a biotechnological point of view. We also highlight recent accomplishments with diverse expression systems and presents different affinity techniques for the capture of recombinant IgA to compare their purification potential.
Topics: Animals; Biotechnology; Humans; Immunoglobulin A; Mice; Models, Molecular; Protein Engineering; Protein Processing, Post-Translational; Recombinant Proteins
PubMed: 25257601
DOI: 10.1007/s10529-014-1686-z