ArticleProtein science : a publication of the Protein Society2002
Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli.
Article in Protein science : a publication of the Protein Society, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 114 papers.
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114 citing papers in PubMed, 333 citations in OpenAlex.
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- Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.bioRxiv : the preprint server for biology · 2024Article
- Expression of the Antimicrobial Peptide SE-33-A2P, a Modified Analog of Cathelicidin, and an Analysis of Its Properties.Antibiotics (Basel, Switzerland) · 2024Article
- Tagging Recombinant Proteins to Enhance Solubility and Aid Purification.Methods in molecular biology (Clifton, N.J.) · 2023Article
- The future of recombinant host defense peptides.Microbial cell factories · 2022Review
- Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli.Microbial cell factories · 2022Article
- Cloning and Characterization of Two NovelInternational journal of molecular sciences · 2022Article
- Protein and antibody purification followed by immunoprecipitation of MYB and GATA zinc finger-type maize proteins with magnetic beads.STAR protocols · 2022Article
- Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases.PLoS pathogens · 2022Article
- Overcoming the Solubility Problem in E. coli: Available Approaches for Recombinant Protein Production.Methods in molecular biology (Clifton, N.J.) · 2022Review
- A Novel Approach for the Production of Aggregation-Prone Proteins Using the Spidroin-Derived NT* Tag.Methods in molecular biology (Clifton, N.J.) · 2022Article
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- A novel approach of recombinant laterosporulin production using the N-SH2 domain of SHP-2.BMC biotechnology · 2021Article
- Strategies for the Production of Soluble Interferon-AlphaLife (Basel, Switzerland) · 2021Article
54 more citing papers are in PubMed but not listed here.
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Authors and funding
5 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.
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