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1.
Dr. Shunsuke Kato Koki Takeuchi Motonao Iwaki Prof. Dr. Kentaro Miyazaki Prof. Dr. Kohsuke Honda Prof. Dr. Takashi Hayashi 《Angewandte Chemie (International ed. in English)》2023,62(31):e202303764
Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction. 相似文献
2.
Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations. 相似文献
3.
Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids
Affinity tags are efficient tools for protein purification. They allow simple one-step purification of proteins to high purity. However, in some cases the tags cause structural and functional changes in a protein, and need to be removed. Therefore, affinity tags that are readily introduced into proteins with minimal perturbation and have specific affinity for purification are desired. Herein, two metal-chelating amino acids derived from 2,2′-bipyridine and 8-hydroxyquinoline were genetically incorporated into glutathione S-transferase (GST) and the mutant proteins were purified by using the metal ion affinity of the unnatural amino acids. The purification of the GST mutants containing 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) showed that the proteins could be efficiently enriched in Ni–NTA by the metal ion affinity of the unnatural amino acid and purified to excellent purity. This method should be very useful for general protein affinity purification, especially for proteins whose structure or function is affected by affinity tags fused to N- or C-terminals. 相似文献
4.
源于大肠杆菌蛋白的表达、液相色谱复性与纯化新进展 总被引:1,自引:0,他引:1
对近两年来源于大肠杆菌(Escherichia coli,E.coli)的蛋白表达和用蛋白折叠液相色谱(protein folding liquid chromatography,PFLC)法对所形成的包涵体目标蛋白的复性并同时纯化的新近发展做了简要的介绍和评述.PFLC法用于包涵体蛋白分离、纯化很广,其特点是除了在色谱柱上将目标蛋白与其他组分分开,还同时要在色谱柱上进行包涵体蛋白折叠.可以说,现代生物技术中所用的大多数有价值蛋白产品的制备仍然有赖于不同机理的液相色谱(Lc)法.而用PFLC法对源于E.coli的蛋白的制备方法更具可塑性和容易达到规模化,其生成本可以成倍地降低.该文主要内容包括了E.coli蛋白的表达及样品前处理、PFLC的实用范围、PFLC的优化、PFLC中的新技术、新设备和新方法、PFLC的分子学机理、应用事例及对未来的展望. 相似文献
5.
Gradiflow is new technology allowing purification of important blood proteins from viral contaminated plasma. Protein purification is based on unique scalable tangential-flow preparative electrophoresis, and is distinct from current technology because protein purification and virus removal are performed in the same step. This one-step removal and purification exploits both the size and charge of target proteins. The medically important blood proteins, immunoglobulin G (IgG) and alpha-1-antitrypsin, were chosen to demonstrate the ability of this process to purify proteins from contaminated plasma. Clearance factors achieved by infectivity assays and polymerase chain reaction (PCR) that meet regulatory requirements demonstrated removal of canine parvovirus (CPV). CPV is a model virus for pathogenic nonenveloped viruses, including parvovirus B19, not adequately removed or inactivated by most processes currently in practice. The recovery of proteins from plasma with high purity, recovery, and function, while simultaneously removing viruses, provides blood products with a level of purity compatible with clinical use more quickly and cheaply than available techniques. 相似文献
6.
嗜硫色谱在高盐环境下对抗体及其他某些蛋白质产生特异性吸附,再在低盐条件下洗脱,可获得高纯度及高回收率的蛋白质产品,是一种新型蛋白质纯化技术。该文对嗜硫色谱及其在抗体纯化中的应用做了综述。 相似文献
7.
《Analytical letters》2012,45(3):407-415
Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based affinity purification for His-tagged proteins for comparison of purification efficiency with the conventional Ni2+-based affinity chromatography. Thiol-functionalized aptamers able to specifically bind to His-tag were immobilized employing two crosslinking methods onto the surface of polystyrene resins. The resulting aptamer-anchored resins were successfully applied for purification of His-tagged proteins from complex E. coli and human cell lysates, respectively, and superior or at least comparable purification results to the conventional immobilized metal affinity chromatography were obtained via one-step purification. 相似文献
8.
Use of a urea and guanidine-HCl-propanol solvent system to purify a growth inhibitory glycopeptide by high-performance liquid chromatography 总被引:1,自引:0,他引:1
Reversed-phase high-performance liquid chromatography was used to purify an inhibitory glycopeptide where resolution and recovery were enhanced by using urea or guanidine-HCl-isopropanol-water as a solvent system. Isopropanol alone or other solvent systems that have been proposed for such purification steps were not effective in eluting hydrophobic proteins from the reversed-phase column. The application of the urea or guanidine-HCl solvent systems in the separation and purification of membrane proteins, and other hydrophobic macromolecules, could greatly enhance recovery and efficiency of purification. 相似文献
9.
Bernhard OK Cunningham AL Sheil MM 《Journal of the American Society for Mass Spectrometry》2004,15(4):558-567
Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies. 相似文献
10.
A red fluorescent protein, DsRed, which emits fluorescence in the red region of the spectrum has become a popular alternative
to green fluorescent protein as a label in biochemical and bioanalytical applications. In this study, we have developed a
simple purification method for DsRed variants utilizing their inherent copper binding property. A purification procedure was
developed and optimized using immobilized copper ions yielding a single strong band corresponding to purified DsRed proteins
on the SDS-PAGE gel. A purification efficiency of higher than 95% was achieved. A spectral analysis and copper binding study
was performed to verify activity of the purified proteins. The development of this method allows DsRed to play a dual role
as a fluorescent reporter protein and as a purification affinity tag for a target protein. This simpler approach of purification
should expand the utility of DsRed. 相似文献
11.
Membrane proteins play essential roles in regulating various fundamental cellular functions. To investigate membrane proteins, extraction and purification are usually prerequisite steps. Here, we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract, which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows, resulting in significantly increased extraction speed and efficiency. To evaluate this system, different separation and detection methods were used to identify the purified proteins, such as capillary electrophoresis, SDS-PAGE and nano-HPLC-MS/MS. Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE. Results indicated that efficient purification of membrane proteins can be achieved within 5-7s and approximately 90% of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date), including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains. Compared to conventional approaches, this new method had advantages of greater specific surface area, minimal emulsification, reduced sample consumption and analysis time. We expect the developed method to be potentially useful in membrane protein purifications, facilitating the investigation of membrane proteomics. 相似文献
12.
Charlotte Cabanne Jérôme PezziniGilles Joucla Agnès HocquelletCaroline Barbot Bertrand GarbayXavier Santarelli 《Journal of chromatography. A》2009,1216(20):4451-4456
Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50–60% range for affinity resins, and in the 80–85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein. 相似文献
13.
Yang P Marinakos SM Chilkoti A 《Langmuir : the ACS journal of surfaces and colloids》2011,27(4):1463-1471
Protein immobilization on surfaces is useful in many areas of research, including biological characterization, antibody purification, and clinical diagnostics. A critical limitation in the development of protein microarrays and heterogeneous protein-based assays is the enormous amount of work and associated costs in the purification of proteins prior to their immobilization onto a surface. Methods to address this problem would simplify the development of interfacial diagnostics that use a protein as the recognition element. Herein, we describe an approach for the facile, site-specific immobilization of proteins on a surface without any preprocessing or sample purification steps that ligates an intein fusion protein at its C-terminus by reaction with a hydrazine group presented by a surface. Furthermore, we demonstrate that this methodology can directly immobilize a protein directly from cell lysate onto a protein-resistant surface. This methodology is also compatible with soft lithography and inkjet printing so that one or more proteins can be patterned on a surface without the need for purification. 相似文献
14.
Dae-Hyukkweon Kweon Sung-Gunkim Kim Jin-Hoseo Seo 《Journal of inclusion phenomena and macrocyclic chemistry》2004,50(1-2):37-41
Recombinant DNA technology and protein engineering are currently utilized in the cost-effective production of pharmaceutical and industrial proteins with native conformation. Escherichia coli retains its dominant position as the first choice of host for speed, simplicity and well-established production protocols. However, protein production using recombinant E. coli occasionally encounters complex purification and refolding steps. This paper introduces an efficient scheme for purification andin vitro refolding of industrially important proteins including cyclodextrin glycosyltransferase (CGTase) expressed in recombinant E. coli employing a polycationic amino acid fusion system. Fusion of polycationic amino acids to CGTase allowed purification and refolding of CGTase to be simple and efficient. A novel CGTase production strategy will be discussed by considering recent progress in protein purification and refolding techniques. 相似文献
15.
A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles 总被引:1,自引:0,他引:1
A novel protein purification method was developed using microbial polyhydroxyalkanoates (PHA) granule-associated protein phasin, a pH-inducible self-cleaving intein and PHA nanoparticles. Genes for the target proteins to be produced and purified were fused to genes of intein and phasin, the genes were jointly over-expressed in vivo, such as in E. coli cells in this study. The fused proteins containing target protein, intein and phasin produced by the recombinant E. coli were released together with all other E. coli proteins via a bacterial lysis process. They were then adsorbed in vitro to the surfaces of the hydrophobic polymer nanoparticles incubated with the cell lysates. The nanoparticles attached with the fused proteins were concentrated via centrifugation. Then, the reasonably purified target protein was released by self-cleavage of intein and separated with nanoparticles by a simple centrifugation process. Using this system, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and beta-galactosidase were successfully purified in their active forms with reasonable yields, respectively, demonstrating the effectiveness and reliability of this purification system. This method allows the production and purification of high value added proteins in a continuous way with low cost. 相似文献
16.
The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification. 相似文献
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A positively charged protein domain, Z(basic), can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Z(basic), was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Z(basic) as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Z(basic) tag in small and pilot-plant-scale downstream processing. 相似文献
20.
《Trends in analytical chemistry : TRAC》1986,5(9):225-230
The three membrane proteins of Sendai virus can be extracted from the membrane by non-ionic detergents. The extracted proteins serve as a model mixture for the development of high-performance liquid chromatographic methods for the purification of hydrophobic membrane proteins. 相似文献