ArticleProtein expression and purification2008
Overexpression of post-translationally modified peptides in Escherichia coli by co-expression with modifying enzymes.
Article in Protein expression and purification, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers.
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Who cites it
24 citing papers in PubMed, 35 citations in OpenAlex.
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- Linker Length and Composition within Disordered Binding Motifs Modulates the Avidity and Reversibility of a Multivalent Protein Interaction Switch.Journal of molecular biology · 2025Article
- Induced Proximity Approach Enables the Recombinant Production of Polyphosphorylated Silk Proteins with Improved Adhesiveness.Biomacromolecules · 2025Article
- Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana.Scientific reports · 2024Article
- The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants.Frontiers in plant science · 2022Article
- The molecular basis of allostery in a facilitated dissociation process.Structure (London, England : 1993) · 2021Article
- Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.Biophysical journal · 2017Article
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- The helical propensity of the extracellular loop is responsible for the substrate specificity of Fe(III)-phytosiderophore transporters.FEBS letters · 2016Article
- The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation.Proceedings of the National Academy of Sciences of the United States of America · 2016Article
- Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.Molecular & cellular proteomics : MCP · 2015Article
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- The high-risk HPV16 E7 oncoprotein mediates interaction between the transcriptional coactivator CBP and the retinoblastoma protein pRb.Journal of molecular biology · 2014Article
- Combinatorial regulation of a signal-dependent activator by phosphorylation and acetylation.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
- Structural basis for inhibition of the MDM2:p53 interaction by an optimized MDM2-binding peptide selected with mRNA display.PloS one · 2014Article
- The arginine-rich RNA-binding motif of HIV-1 Rev is intrinsically disordered and folds upon RRE binding.Biophysical journal · 2013Article
- Modified recombinant proteins can be exported via the Sec pathway in Escherichia coli.PloS one · 2012Article
- Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy.Journal of biomolecular NMR · 2011Article
- Production of active recombinant eIF5A: reconstitution in E.coli of eukaryotic hypusine modification of eIF5A by its coexpression with modifying enzymes.Protein engineering, design & selection : PEDS · 2011Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
Post-translational modification plays crucial roles in signal transduction in eukaryotic cells. To elucidate the biological function of a protein with a specific post-translational modification, it is necessary to isolate the modified protein. However, it is difficult to incorporate a modified amino acid into a specific position of a protein, in particular, in a large-scale preparation. In order to prepare post-translationally modified proteins in Escherichia coli (E. coli), we have constructed co-expression vectors that contain protein and corresponding enzyme genes. The protein and enzyme are co-expressed in the same E. coli cells and the protein is post-translationally modified in vivo. By using this system, the transcriptional activator cyclic-AMP-response-element-binding protein (CREB) was phosphorylated at Ser-133 and the hypoxia-inducible factor-1alpha (HIF-1alpha) was hydroxylated at Asn-803 in E. coli. Although the constructs of the proteins we used are very flexible and susceptible to degradation by proteases in E. coli when they are expressed alone, the B1 domain of streptococcal protein G (GB1) fused to the N-terminus of the proteins increased the yields dramatically. Site-specific phosphorylation of CREB and hydroxylation of HIF-1alpha were confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and NMR. Our GB1-fusion co-expression system can be used in the same way as conventional protein expression in E. coli, making it a flexible and economical method to produce a large amount of a post-translationally modified protein.
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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.