ArticleProtein expression and purification2009
A simple and efficient expression and purification system using two newly constructed vectors.
Article in Protein expression and purification, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 79 papers.
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Who cites it
79 citing papers in PubMed, 130 citations in OpenAlex.
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- Non-canonical binding of the Chaetomium thermophilum PolD4 N-terminal PIP motif to PCNA involves Q-pocket and compact 2-fork plug interactions but no 3The FEBS journal · 2023Article
- Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping.Methods in molecular biology (Clifton, N.J.) · 2022Article
- A new catalytic mechanism of bacterial ferredoxin-NADPProtein science : a publication of the Protein Society · 2021Article
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- The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes.Nature chemical biology · 2020Article
- Corrigendum: Pac13 is a Small Dehydratase that Mediates the Formation of the 3'-Deoxy Nucleoside of Pacidamycins.Angewandte Chemie (International ed. in English) · 2020Article
- Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Low Level Antibodies Against Alpha-Tropomyosin Are Associated With Increased Risk of Coronary Heart Disease.Frontiers in pharmacology · 2020Article
- Identification of the First Gene Transfer Agent (GTA) Small Terminase inJournal of virology · 2019Article
- Elucidation of a sialic acid metabolism pathway in mucus-foraging Ruminococcus gnavus unravels mechanisms of bacterial adaptation to the gut.Nature microbiology · 2019Article
19 more citing papers are in PubMed but not listed here.
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility screening and purification efficiency. The system is based on two newly constructed vectors, pEHISTEV and pEHISGFPTEV derived from a pET vector. Both vectors generate a construct with an amino-terminal hexahistidine tag (His-tag). In addition, pEHISGFPTEV expresses a protein with an N-terminal His-tagged green fluorescent protein (GFP) fusion to allow rapid quantitation of soluble protein. Both vectors have a tobacco etch virus (TEV) protease cleavage site that allows for production of protein with only two additional N-terminal residues and have the same multiple cloning site which enables parallel cloning. Protein purification is a simple two-stage nickel affinity chromatography based on the His tag removal. A total of seven genes were tested using this system. Expression was optimised using pEHISGFPTEV constructs by monitoring the GFP fluorescence and the soluble target proteins were quantified using spectrophotometric analysis. All the tested proteins were purified with sufficient quantity and quality to attempt structure determination. This system has been proven to be simple and effective for structural biology. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.