ArticleThe Biochemical journal1999
Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein.
Article in The Biochemical journal, 1999. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 61 citations in OpenAlex.
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- Expression, purification and structural properties of ABC transporter ABCA4 and its individual domains.Protein expression and purification · 2014Article
- Several affinity tags commonly used in chromatographic purification.Journal of analytical methods in chemistry · 2013Review
- The use of Bcl-2 over-expression to stabilize hybridomas specific to the HERG potassium channel.Journal of immunological methods · 2012Article
- An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production.Journal of industrial microbiology & biotechnology · 2011Article
- Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii.Plant biotechnology journal · 2010Article
- Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli.Virology journal · 2010Article
- Mining mammalian genomes for folding competent proteins using Tat-dependent genetic selection in Escherichia coli.Protein science : a publication of the Protein Society · 2009Article
- The NBDs that wouldn't die: A cautionary tale of the use of isolated nucleotide binding domains of ABC transporters.Communicative & integrative biology · 2009Article
- Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO.Protein science : a publication of the Protein Society · 2006Article
- Site-directed mutagenesis and expression of the soluble form of the family IIIa cellulose binding domain from the cellulosomal scaffolding protein of Clostridium cellulovorans.Journal of bacteriology · 2005Article
- SUMO fusion technology for difficult-to-express proteins.Protein expression and purification · 2005Review
- Nucleotides and phospholipids compete for binding to the C terminus of KATP channels.Proceedings of the National Academy of Sciences of the United States of America · 2002Article
- Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli.Protein science : a publication of the Protein Society · 2002Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.
Indexed as
Identifiers
9931301PMC1220027W2018417316What OpenQuestion holds
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.