Conditional protein splicing of α-sarcin in live cells

Alford, Spencer C.; O’Sullivan, Connor; Obst, Jon K; Christie, Jennifer; Howard, Perry L.

doi:10.1039/c3mb70387h

Cited by 10 publications

(5 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, there are some exceptions to this rule. For example, the artificially split Sce VMA intein can undergo PTS without a refolding step if (and only if) the two fragments, VMA N and VMA C , are brought into proximity using a two- or three-hybrid paradigm (Figure b). − In another example, Mootz and co-workers demonstrated that the artificially split Ssp DnaB intein can effect PTS under native conditions, albeit with reduced efficiency compared to the parental contiguous intein (Figure c) …”

Section: Protein Trans-splicingmentioning

confidence: 99%

Chemoenzymatic Semisynthesis of Proteins

2019

View full text Add to dashboard Cite

Protein semisynthesisdefined herein as the assembly of a protein from a combination of synthetic and recombinant fragmentsis a burgeoning field of chemical biology that has impacted many areas in the life sciences. In this review, we provide a comprehensive survey of this area. We begin by discussing the various chemical and enzymatic methods now available for the manufacture of custom proteins containing noncoded elements. This section begins with a discussion of methods that are more chemical in origin and ends with those that employ biocatalysts. We also illustrate the commonalities that exist between these seemingly disparate methods and show how this is allowing for the development of integrated chemoenzymatic methods. This methodology discussion provides the technical foundation for the second part of the review where we cover the great many biological problems that have now been addressed using these tools. Finally, we end the piece with a short discussion on the frontiers of the field and the opportunities available for the future.

show abstract

Section: Protein Trans-splicingmentioning

confidence: 99%

Chemoenzymatic Semisynthesis of Proteins

2019

View full text Add to dashboard Cite

show abstract

“…Close proximity then allows the inteins to fold and activate splicing ( Figure 1B). This approach, for which the term 'conditional protein splicing (CPS)' was actually coined, was applied in a variety of ways, for example in vitro for the control of an artificially autoinhibited protein kinase (Mootz et al, 2004), to activate tobacco etch virus (TEV) protease in yeast cells (Sonntag and Mootz, 2011), to induce apoptosis in mammalian cells via the RNAse α-sarcin (Alford et al, 2014) and to regulate firefly luciferase in living fruit flies, which showed detectable luciferase activity as early as 10 to 20 min after exposure to rapamycin-containing food (Schwartz et al, 2007) (see Table 1 for more examples of ligand-controlled Sce VMA intein).…”

Section: Methods To Control Split Inteinsmentioning

confidence: 99%

Switchable inteins for conditional protein splicing

Ventura

Mootz

2018

Biological Chemistry

View full text Add to dashboard Cite

Synthetic biologists aim at engineering controllable biological parts such as DNA, RNA and proteins in order to steer biological activities using external inputs. Proteins can be controlled in several ways, for instance by regulating the expression of their encoding genes with small molecules or light. However, post-translationally modifying pre-existing proteins to regulate their function or localization leads to faster responses. Conditional splicing of internal protein domains, termed inteins, is an attractive methodology for this purpose. Here we discuss methods to control intein activity with a focus on those compatible with applications in living cells.

show abstract

“…The reporter protein functions can be inducibly controlled by enzymes, [6] chemical compounds, [7] ions, [8] light, [9] and so on. To this end, inteins, which are single‐turnover enzymes catalyzing protein splicing, [10] have been used [11–15] . Naturally occurring inteins are categorized into three groups: inteins that have a homing endonuclease between the splicing domains, inteins that have only a splicing domain, and inteins that catalyze splicing in trans (protein trans ‐splicing; PTS) through split inteins.…”

Section: Introductionmentioning

confidence: 99%

Activation of Protease and Luciferase Using Engineered Nostoc punctiforme PCC73102 DnaE Intein with Altered Split Position

2020

View full text Add to dashboard Cite

Inteins, self‐catalytic enzymes, have been widely used in the field of protein engineering and chemical biology. Here, Nostoc punctiforme PCC73102 (Npu) DnaE intein was engineered to have an altered split position. An 11‐residue N‐intein of DnaE in which Gly and Asp were substituted for Tyr4 and Glu5, respectively, was designed, and the active C‐intein variants were acquired by a GFP fluorescence‐based screening. The designed N‐intein and the obtained active C‐intein variants were used to construct a turn‐on system for enzyme activities such as human immunodeficiency 1 protease and NanoLuc luciferase. Based on the NanoLuc‐intein fusion, we developed two intein pairs, each of which is capable of reacting preferentially, by interchanging the charged amino acids on N‐ and C‐inteins. The specific splicing reactions were easily monitored and discriminated by bioluminescence resonance energy transfer (BRET).

show abstract

Conditional protein splicing of α-sarcin in live cells

Cited by 10 publications

References 41 publications

Chemoenzymatic Semisynthesis of Proteins

Chemoenzymatic Semisynthesis of Proteins

Switchable inteins for conditional protein splicing

Activation of Protease and Luciferase Using Engineered Nostoc punctiforme PCC73102 DnaE Intein with Altered Split Position

Contact Info

Product

Resources

About