The Experts below are selected from a list of 104931 Experts worldwide ranked by ideXlab platform
Guo-qiang Chen - One of the best experts on this subject based on the ideXlab platform.
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Microbial polyhydroxyalkanote synthesis repression Protein PhaR as an affinity tag for recombinant Protein Purification
Microbial cell factories, 2010Co-Authors: Shuang Zhang, Zhi Hui Wang, Guo-qiang ChenAbstract:Background PhaR which is a repressor Protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro Protein Purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target Protein allowed Protein Purification with a pH and temperature shift. During the process, the target Protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.
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repression Protein PhaR as an affinity tag for recombinant Protein Purification
2010Co-Authors: Shuang Zhang, Zhi Hui Wang, Guo-qiang ChenAbstract:Background: PhaR which is a repressor Protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro Protein Purification. Fusion of PhaRtagged self-cleavable Ssp DnaB intein to the N-terminus of a target Protein allowed Protein Purification with a pH and temperature shift. During the process, the target Protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation. Results: Fusion Protein PhaR-intein-target Protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target Protein was released from the particles and purified after centrifugation. As target Proteins, enhanced green fluorescent Protein (EGFP), maltose binding Protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based Protein Purification method. Conclusion: The successful Purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro Purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple Protein Purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial Protein Purification systems.
Sophia Hober - One of the best experts on this subject based on the ideXlab platform.
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An Orthogonal Fusion Tag for Efficient Protein Purification.
Methods in molecular biology (Clifton N.J.), 2020Co-Authors: Johan Nilvebrant, Mikael Åstrand, Sophia HoberAbstract:In this chapter, we present an efficient method for stringent Protein Purification facilitated by a dual affinity tag referred to as ABDz1, which is based on a 5 kDa albumin-binding domain from Streptococcal Protein G. The small fusion tag enables an orthogonal affinity Purification approach based on two successive and highly specific affinity Purification steps. This approach is enabled by native binding of ABDz1 to human serum albumin and engineered binding to Staphylococcal Protein A, respectively. The ABDz1-tag can be fused to either terminus of a Protein of interest and the Purification steps can be carried out using standard laboratory equipment.
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An orthogonal fusion tag for efficient Protein Purification.
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Johan Nilvebrant, Mikael Åstrand, Sophia HoberAbstract:Protein fusion tags are important tools in research when robust methods for Protein Purification and detection are required. In this chapter we present an efficient method for stringent Protein Purification. A small domain, denoted ABDz1, with affinity for both human serum albumin and Protein A has been developed. The Purification tag is based on an albumin-binding domain from Streptococcal Protein G that was engineered to bind Protein A. The ABDz1-tag can be fused to any Protein of choice and the Purification can be performed using standard laboratory equipment. In this chapter a method for Purification of ABDz1-tagged Proteins using two successive affinity Purification steps is described.
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High-throughput Protein Purification using an automated set-up for high-yield affinity chromatography.
Protein expression and purification, 2006Co-Authors: Johanna Steen, Sophia Hober, Mathias Uhlén, Jenny OttossonAbstract:One of the key steps in high-throughput Protein production is Protein Purification. A newly developed high-yield Protein Purification and isolation method for laboratory scale use is presented. This procedure allows fully automated Purification of up to 60 cell lysates with milligram yields of pure recombinant Protein in 18.5 h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput Protein Purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high Protein yield and purity.
Harm Jan Arjan Snijder - One of the best experts on this subject based on the ideXlab platform.
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A fast and easy strategy for Protein Purification using "teabags".
Scientific reports, 2016Co-Authors: Marie Castaldo, L. Barlind, F. Mauritzson, P. T. Wan, Harm Jan Arjan SnijderAbstract:Protein Purification often involves affinity capture of Proteins on stationary resin, alternatively Proteins are captured on free flowing resin for subsequent separation from bulk fluid. Both methods require labour and time intensive separation of particulate matter from fluid. We present a method where affinity resin is contained within porous-walled containers, supporting clarification, product recovery, and concentration in a single step with minimal hands-on processing time, without significant investments in equipment.
Shuang Zhang - One of the best experts on this subject based on the ideXlab platform.
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Microbial polyhydroxyalkanote synthesis repression Protein PhaR as an affinity tag for recombinant Protein Purification
Microbial cell factories, 2010Co-Authors: Shuang Zhang, Zhi Hui Wang, Guo-qiang ChenAbstract:Background PhaR which is a repressor Protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro Protein Purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target Protein allowed Protein Purification with a pH and temperature shift. During the process, the target Protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.
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repression Protein PhaR as an affinity tag for recombinant Protein Purification
2010Co-Authors: Shuang Zhang, Zhi Hui Wang, Guo-qiang ChenAbstract:Background: PhaR which is a repressor Protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro Protein Purification. Fusion of PhaRtagged self-cleavable Ssp DnaB intein to the N-terminus of a target Protein allowed Protein Purification with a pH and temperature shift. During the process, the target Protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation. Results: Fusion Protein PhaR-intein-target Protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target Protein was released from the particles and purified after centrifugation. As target Proteins, enhanced green fluorescent Protein (EGFP), maltose binding Protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based Protein Purification method. Conclusion: The successful Purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro Purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple Protein Purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial Protein Purification systems.
David W. Wood - One of the best experts on this subject based on the ideXlab platform.
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Encyclopedia of Industrial Biotechnology - Large‐Scale Protein Purification, Self‐Cleaving Aggregation Tags
Encyclopedia of Industrial Biotechnology, 2010Co-Authors: Iraj Ghazi, David W. WoodAbstract:In the mid-1980s, the incorporation of affinity tags into recombinant Proteins by means of genetic engineering created a highly efficient tool for Protein Purification. A recent advance in this area has been the addition of self-cleaving Protein domains to conventional affinity tags, which allows the tags to self-cleave and thus eliminates the requirement for proteolytic removal of the tag. In practice, the native target is released from the affinity tag after Purification by a simple temperature, chemical, and/or pH change. Although this is a significant advance for affinity-tag methods, some limitations still arise from the equipment and consumable resin costs associated with these procedures. In this contribution, we describe solutions to these limitations through the development of a new generation of self-associating and aggregating tags. One of these tags uses the association of phasin Proteins to polyhydroxybutyrate granules coexpressed in the host cell, producing a system where the host cell also produces the affinity matrix. The other is based on reversibly precipitating elastin-like polypeptide tags that can be separated from cellular contaminants by simple heating and centrifugation. Both these tags allow Protein Purification to take place using simple mechanical methods, thus eliminating the need for conventional affinity apparatus or reagents. Self-cleaving aggregation-tag technologies may dramatically reduce recombinant Protein Purification costs. Keywords: affinity tag; aggregation tag; bioseparation; intein;, recombinant Protein Purification; self-cleaving tag
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Inteins and affinity resin substitutes for Protein Purification and scale up
Microbial Cell Factories, 2005Co-Authors: Mahmoud Reza Banki, David W. WoodAbstract:The development of self-cleaving fusion-tag technology has greatly simplified the Purification of recombinant Proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional Purification tags to generate novel and convenient Protein Purification methods at a variety of scales. In this review, we describe some of these methods, and provide a rudimentary economic analysis of hypothetical large-scale applications. This work is expected to provide a rough outline for the evaluation of these methods for large-scale bioprocessing of a variety of products.
