ArticleProtein science : a publication of the Protein Society1999
Intrabody construction and expression III: engineering hyperstable V(H) domains.
Article in Protein science : a publication of the Protein Society, 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.
- A blinded, prospective benchmark of in silico antibody discovery anchored to experimental affinity and developability.Nature biotechnology · 2026Article
- Article
- Designer Oncolytic Adenovirus: Coming of Age.Cancers · 2018Review
- Consensus protein design.Protein engineering, design & selection : PEDS · 2016Review
- Protein stability: computation, sequence statistics, and new experimental methods.Current opinion in structural biology · 2015Review
- Advances in Antibody Design.Annual review of biomedical engineering · 2015Review
- Protein stability by number: high-throughput and statistical approaches to one of protein science's most difficult problems.Current opinion in chemical biology · 2011Review
- Article
- Intrabody and Parkinson's disease.Biochimica et biophysica acta · 2009Review
- Energetics of aliphatic deletions in protein cores.Protein science : a publication of the Protein Society · 2006Article
- Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation.The EMBO journal · 2003Article
- De novo production of diverse intracellular antibody libraries.Nucleic acids research · 2003Article
- Functional aspects of co-variant surface charges in an antibody fragment.Protein science : a publication of the Protein Society · 2002Article
- The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis.Protein science : a publication of the Protein Society · 2000Article
- Article
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2 authors.
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No grant is acknowledged in the PubMed record.
Abstract
The folding of immunoglobulin domains requires the formation of a conserved structural disulfide. Therefore, as a general rule, they cannot be functionally expressed in the reducing environment of the cellular cytoplasm. We have previously reported that stability engineering can lead to the cytoplasmic expression of functional immunoglobulin V(L) domains. Here we apply rational stability engineering by consensus sequence analysis to V(H) domains. Isolated V(H) domains tend to aggregate more easily than V(L) domains; they do not refold quantitatively and are generally more difficult to handle in vitro. To overcome these problems, we successfully predicted and experimentally verified several stabilizing point mutations in the V(H) domain of a designed, catalytic Fv fragment. The effect of single mutations was additive, and they could be combined in a prototype domain with significantly improved stability against chemical denaturation and a 20-fold increased half time of irreversible thermal denaturation, at physiological temperature. This stabilized, isolated V(H) domain could be expressed solubly in the reducing cellular cytoplasm of Escherichia coli, at a yield of approximately 1.2 mg/L of shake flask culture. It remains fully functional, as evidenced by the successful reconstitution of an esterolytic Fv fragment with the V(L) domain. This success provides further evidence that consensus sequence engineering is a rational, plannable route to the construction of intrabodies.
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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.