Article

A designed protein for the specific and covalent heteroconjugation of biomolecules

Ecole Polytechnique Fédérale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering, CH-1015 Lausanne, Switzerland.
Bioconjugate Chemistry (Impact Factor: 4.82). 09/2008; 19(9):1753-6. DOI: 10.1021/bc800268j
Source: PubMed

ABSTRACT Bioconjugations often rely on adaptor molecules to cross-link different biomolecules. In this work, we introduce the molecular adaptor covalin, which is a protein chimera of two self-labeling proteins with nonoverlapping substrate specificity. Covalin permits a selective and covalent heteroconjugation of biomolecules displaying appropriate functional groups. Examples for the use of covalin include the specific heteroconjugation of a reporter enzyme to an antibody and of molecular probes to the surface of living cells. The efficiency and specificity of covalin-based bioconjugations together with the availability of a large variety of substrates create immediate and ubiquitous applications for covalin in bioconjugate chemistry.

0 Followers
 · 
80 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: A series of water-soluble red-emitting distyryl-borondipyrromethene (BODIPY) dyes were designed and synthesized by using three complementary approaches aimed at introducing water-solubilizing groups on opposite faces of the fluorescent core to reduce or completely suppress self-aggregation. An additional carboxylic acid functional group was introduced at the pseudo-meso position of the BODIPY scaffold for conjugation to amine-containing biomolecules/biopolymers. The optical properties of these dyes were evaluated under simulated physiological conditions (i.e., phosphate-buffered saline (PBS), pH 7.5) or in pure water. The emission wavelength (λ(max)) of these labels was found in the 640-660 nm range with quantum yields from modest to unprecedentedly high values (4 to 38%). The bioconjugation of these distyryl-BODIPY dyes with bovine serum albumin (BSA) and the monoclonal antibody (mAb) 12A5 was successfully performed under mild aqueous conditions.
    Chemistry - A European Journal 06/2012; 18(23):7229-42. DOI:10.1002/chem.201103613 · 5.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bioorthogonal ligation methods that allow the selective conjugation of fluorophores or biotin to proteins and small molecule probes that contain inert chemical handles are an important component of many chemical proteomic strategies. Here, we present a new catch-and-release enrichment strategy that utilizes a hexylchloride group as a bioorthogonal chemical handle. Proteins and small molecules that contain a hexylchloride tag can be efficiently captured by an immobilized version of the self-labeling protein HaloTag. Furthermore, by using a HaloTag fusion protein that contains a protease cleavage site, captured proteins can be selectively eluted under mild conditions. We demonstrate the utility of the hexylchloride-based catch-and-release strategy by enriching protein kinases that are covalently and non-covalently bound to ATP-binding site-directed probes from mammalian cell lysates. Our catch-and-release system creates new possibilities for profiling enzyme families and for the identification of the cellular targets of bioactive small molecules.
    ACS Chemical Biology 01/2013; 8(4). DOI:10.1021/cb300623a · 5.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Enzyme-promoted assembly: The construction of a hetero-bifunctional protein building block, HaloTag-cutinase, that reacts rapidly and selectively with a small-molecule linker is described. The step-wise combination of these building blocks generates a 300 kDa "megamolecule" with precisely defined domain orientation, connectivity, and composition.
    ChemBioChem 11/2012; 13(16). DOI:10.1002/cbic.201200501 · 3.06 Impact Factor