Temperature-Sensitive Mutations Made Easy: Generating Conditional Mutations by Using Temperature-Sensitive Inteins That Function Within Different Temperature Ranges

Tan, Guihong; Chen, Ming; Foote, Christopher; Tan, Change

doi:10.1534/genetics.109.104794

Cited by 45 publications

(39 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Particularly to optimize cleavage efficiency, inteins have been mutagenized to become inducible by pH (21), reducing agents (22), or temperature (23) and to separate cleavage activity from splicing. Interestingly, inteins can be engineered to cut either at the amino terminus (22) or at the carboxyl terminus (24).…”

mentioning

confidence: 99%

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Carvajal-Vallejos

Pallisse

Mootz

et al. 2012

Journal of Biological Chemistry

150

186

View full text Add to dashboard Cite

Background: Hypothetical natural split inteins from environmental metagenomic sources were evaluated experimentally for the first time. Results: Four inteins showed the highest known reaction rates and efficiencies for protein trans-splicing and could be used efficiently for C-cleavage. Conclusion: These inteins pushed the catalytic limit of protein trans-splicing, although this was unpredictable from their primary sequence. Significance: These inteins hold great potential as versatile protein engineering tools.

show abstract

mentioning

confidence: 99%

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Carvajal-Vallejos

Pallisse

Mootz

et al. 2012

Journal of Biological Chemistry

150

186

View full text Add to dashboard Cite

show abstract

“…Directed molecular evolution (DME) of inteins has been shown to be a feasible undertaking, and many cis-splicing inteins have been improved and advanced towards specific reaction conditions (112)(113)(114)(115); an experimental platform for the in vitro DME of split inteins has also been reported recently (116). It remains to be seen whether a unique 'super intein', which combines all favorable characteristics, can be developed through DME, or if the solution will lie in the identification of a set of inteins, each of them suitable for a specific experimental purpose.…”

Section: Outlook: the Future Of Protein Trans-splicingmentioning

confidence: 99%

Protein trans-splicing as a protein ligation tool to study protein structure and function

Aranko

Volkmann²

2011

BioMolecular Concepts

View full text Add to dashboard Cite

Protein trans-splicing (PTS) exerted by split inteins is a protein ligation reaction which enables overcoming the barriers of conventional heterologous protein production. We provide an overview of the current state-of-the-art in split intein engineering, as well as the achievements of PTS technology in the realm of protein structure-function analyses, including incorporation of natural and artificial protein modifications, controllable protein reconstitution, segmental isotope labeling and protein cyclization. We further discuss factors crucial for the successful implementation of PTS in these protein engineering approaches, and speculate on necessary future endeavours to make PTS a universally applicable protein ligation tool.

show abstract

“…These include rapid response, reversibility, and applicability to all tissues and stages of the life cycle of the organism. Although there are tools and techniques for designing ts mutants (34)(35)(36), there have been few systematic studies on the rational or semirational generation of cs mutants of a protein (12,13). We propose a simple and effective technique for generating cs phenotypes by selectively modulating the expression levels of rationally designed partial loss-of-function mutants.…”

mentioning

confidence: 99%

Rational elicitation of cold-sensitive phenotypes

Baliga

Majhi

Mondal

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.conditional mutants | rational design | cold sensitivity | heat-induced expression | transfer between organisms C onditional mutants are powerful tools for studying gene function in vivo. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and lacks that function under a different set of conditions, called restrictive, whereas the wild type (WT) phenotype is similar across both conditions. Cold-sensitive (cs) mutants behave like loss-of-function mutants at temperatures lower than a cutoff temperature, but have WT-like phenotypes at higher temperatures. In contrast, temperature-sensitive (ts) mutants show heat sensitivity and behave like the WT below the restrictive temperature. Both cs and ts mutants can provide important information about protein structure, function, and assembly. cs mutants are rarer than ts mutants, and the molecular basis for generation of cs phenotypes is currently unclear.cs mutants have been used to analyze various biological phenomena, most commonly the cell cycle (1-3), ribosome assembly (4-8), and protein export (9-11), as well as to understand macromolecular structure-function relationships (12, 13). Various ts and cs variants also have helped us understand P22 phage coat protein assembly. Using a combination of both ts and cs mutants causing defects at different stages of the assembly pathway, the order of the various steps occurring along this pathway has been determined (14). Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).Various changes at the amino acid level or gene regulation level can cause cold sensitivity. Some cs mutants have been suggested to result from altered feedback inhibition of certain metabolic pathways at lower temperatures (19,20). Cold sensitivity also has been attribut...

show abstract

Temperature-Sensitive Mutations Made Easy: Generating Conditional Mutations by Using Temperature-Sensitive Inteins That Function Within Different Temperature Ranges

Cited by 45 publications

References 37 publications

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Protein trans-splicing as a protein ligation tool to study protein structure and function

Rational elicitation of cold-sensitive phenotypes

Contact Info

Product

Resources

About