Simultaneous “One Pot” Expressed Protein Ligation and Cu<sup>I</sup>‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization

Steinhagen, Max; Holland‐Nell, Kai; Meldal, Morten; Beck‐Sickinger, Annette G.

doi:10.1002/cbic.201100434

Cited by 16 publications

(12 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Subsequently the azide and alkyne functionalities can be used to immobilize proteins on surfaces since CuAAC reactions require lower concentrations of substrates than does EPL. 6,15,31,32 Thus, by performing the EPL reaction with cysteine azide or cysteine alkyne in solution, high concentrations of the derivatized cysteine can be used to drive efficient functionalization of the proteins.…”

Section: Resultsmentioning

confidence: 99%

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

et al. 2013

View full text Add to dashboard Cite

Intein-mediated expressed protein ligation (EPL) permits the site-specific chemical customization of proteins. While traditional techniques have used purified, soluble proteins, we have extended these methods to release and modify intein fusion proteins expressed on the yeast surface, thereby eliminating the need for soluble protein expression and purification. To this end, we sought to simultaneously release yeast surface-displayed proteins and selectively conjugate with chemical functionalities compatible with EPL and click chemistry. Single-chain antibodies (scFv) and green fluorescent protein (GFP) were displayed on the yeast surface as fusions to the N-terminus of the Mxe GyrA intein. ScFv and GFP were released from the yeast surface with either a sulfur nucleophile (MESNA) or a nitrogen nucleophile (hydrazine) linked to an azido group. The hydrazine azide permitted the simultaneous release and azido functionalization of displayed proteins, but nonspecific reactions with other yeast proteins were detected, and cleavage efficiency was limited. In contrast, MESNA released significantly more protein from the yeast surface while also generating a unique thioester at the carboxy-terminus of the released protein. These protein thioesters were subsequently reacted with a cysteine alkyne in an EPL reaction and then employed in an azide–alkyne cycloaddition to immobilize the scFv and GFP on an azide-decorated surface with >90% site-specificity. Importantly, the immobilized proteins retained their activity. Since yeast surface display is also a protein engineering platform, these approaches provide a particularly powerful tool for the rapid assessment of engineered proteins.

show abstract

Section: Resultsmentioning

confidence: 99%

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

et al. 2013

View full text Add to dashboard Cite

show abstract

“…By performing the CuAAC, SDF-1 could be immobilized and upon specific enzymatic cleavage, subsequently released (Steinhagen et al, 2014). A related study could demonstrate that the CuAAC and the native chemical ligation are orthogonal to each other and can thus be applied in a simultaneous one pot reaction (Steinhagen et al, 2011). Azides can undergo cycloadditions with strained alkynes without the need of a catalyst.…”

Section: Maleimide-thiol Michael Additionmentioning

confidence: 99%

“…Proteins can be absorbed on surfaces by electrostatic, hydrophobic or hydrogen bond interactions. However, the adsorptive immobilization is rather weak, can be easily displaced by other molecules or can alter the activity of the proteins by conformational changes (Steinhagen et al, 2011). Moreover, proteins possess not only properties that beneficially affect cell function.…”

Section: Introductionmentioning

confidence: 99%

Multifunctional biomaterial coatings: synthetic challenges and biological activity

Pagel

Beck‐Sickinger

2017

Biological Chemistry

Self Cite

View full text Add to dashboard Cite

A controlled interaction of materials with their surrounding biological environment is of great interest in many fields. Multifunctional coatings aim to provide simultaneous modulation of several biological signals. They can consist of various combinations of bioactive, and bioinert components as well as of reporter molecules to improve cell-material contacts, prevent infections or to analyze biochemical events on the surface. However, specific immobilization and particular assembly of various active molecules are challenging. Herein, an overview of multifunctional coatings for biomaterials is given, focusing on synthetic strategies and the biological benefits by displaying several motifs.

show abstract

“…Instead of non-natural amino acid incorporation, EPL can facilitate N- or C-terminus-specific functionalization of proteins with azides or alkyne moieties 65 – 67 . The method has been combined with the azide–alkyne cycloaddition, amongst others, to immobilize GFP and aldo-keto reductase on a PEG-based solid support 68 , GFP and single-chain variable fragment antibodies on agarose beads 69 , and nanobodies on polymersome vesicles 70 . A HER2 affibody, a 6.5 kDa protein with high affinity for the oncogene HER2/neu receptor, has been tethered to superparamagnetic iron oxide (SPIO) nanoparticles 71 .…”

Section: Chemical Approaches For Covalent Site-specific Protein Immomentioning

confidence: 99%

Recent advances in covalent, site-specific protein immobilization

Meldal

Schoffelen

2016

F1000Res

Self Cite

View full text Add to dashboard Cite

The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

show abstract

Simultaneous “One Pot” Expressed Protein Ligation and Cu^I‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization

Cited by 16 publications

References 60 publications

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Multifunctional biomaterial coatings: synthetic challenges and biological activity

Recent advances in covalent, site-specific protein immobilization

Contact Info

Product

Resources

About

Simultaneous “One Pot” Expressed Protein Ligation and CuI‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization

Cited by 16 publications

References 60 publications

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Facile Chemical Functionalization of Proteins through Intein-Linked Yeast Display

Multifunctional biomaterial coatings: synthetic challenges and biological activity

Recent advances in covalent, site-specific protein immobilization

Contact Info

Product

Resources

About

Simultaneous “One Pot” Expressed Protein Ligation and Cu^I‐Catalyzed Azide/Alkyne Cycloaddition for Protein Immobilization