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From genes to protein mechanics on a chip.

Otten M, Ott W, Jobst MA, Milles LF, Verdorfer T, Pippig DA, Nash MA, Gaub HE - Nat. Methods (2014)

Bottom Line: Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel.A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever.The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

View Article: PubMed Central - PubMed

Affiliation: 1] Lehrstuhl für Angewandte Physik, Ludwig-Maximilians-Universität, Munich, Germany. [2] Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Munich, Germany. [3].

ABSTRACT
Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

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Method workflow from gene array to single-molecule mechanics.(a) A gene array was spotted onto a glass slide. Genes were designed with a common set of flanking sequences, including a T7 promoter region, a ybbR tag, a Dockerin tag and a T7 terminator. The multilayer microfluidic chip featuring 640 unit cells was aligned to the DNA microarray and bonded to the glass slide. Each unit cell comprised a DNA chamber, a protein chamber, and superseding elastomeric control valves actuated by pneumatic pressure. (b) Control valves were utilized for spatially selective surface modification of each protein chamber with PEG-CoA, and for fluidic isolation of each chamber prior to in vitro expression of the microspotted DNA. Fluorescent labeling with TagRFP-Cohesin was achieved by partial button valve pressurization, leaving only an outer concentric ring of immobilized gene products exposed to the labeling solution. (c) After removal of the microfluidic device, the resulting well-defined, covalently attached protein microarray was accessed from above with a Cohesin-functionalized AFM cantilever. Single-molecule unfolding traces of each of the investigated protein constructs were thus acquired sequentially at each corresponding array address with a single cantilever in a single experiment.
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Figure 1: Method workflow from gene array to single-molecule mechanics.(a) A gene array was spotted onto a glass slide. Genes were designed with a common set of flanking sequences, including a T7 promoter region, a ybbR tag, a Dockerin tag and a T7 terminator. The multilayer microfluidic chip featuring 640 unit cells was aligned to the DNA microarray and bonded to the glass slide. Each unit cell comprised a DNA chamber, a protein chamber, and superseding elastomeric control valves actuated by pneumatic pressure. (b) Control valves were utilized for spatially selective surface modification of each protein chamber with PEG-CoA, and for fluidic isolation of each chamber prior to in vitro expression of the microspotted DNA. Fluorescent labeling with TagRFP-Cohesin was achieved by partial button valve pressurization, leaving only an outer concentric ring of immobilized gene products exposed to the labeling solution. (c) After removal of the microfluidic device, the resulting well-defined, covalently attached protein microarray was accessed from above with a Cohesin-functionalized AFM cantilever. Single-molecule unfolding traces of each of the investigated protein constructs were thus acquired sequentially at each corresponding array address with a single cantilever in a single experiment.

Mentions: Here we developed a platform for parallel characterization of protein mechanics in a single experiment (Fig. 1). Microspotted gene arrays were utilized to synthesize fusion proteins in situ using cell-free gene expression. Proteins were covalently immobilized inside multilayer microfluidic circuits. A single cantilever was then positioned above the protein array, and used to probe the mechanical response of each protein via a common C-terminal Dockerin (Doc) fusion tag. Genes of interest were chosen such that each gene product exhibited an identifiable unfolding pattern when loaded from the N- to C-terminus. Each target protein was expressed with an N-terminal 11 amino acid ybbR tag, which was used to covalently and site-specifically link it to the surface via Sfp Synthase-catalyzed reaction with coenzyme A (CoA)12. At the C-terminus the proteins contained a 75 amino acid cellulosomal Doc from Clostridium thermocellum (C.t.) as specific handle targeted by the Cohesin (Coh)-modified cantilever.


From genes to protein mechanics on a chip.

Otten M, Ott W, Jobst MA, Milles LF, Verdorfer T, Pippig DA, Nash MA, Gaub HE - Nat. Methods (2014)

Method workflow from gene array to single-molecule mechanics.(a) A gene array was spotted onto a glass slide. Genes were designed with a common set of flanking sequences, including a T7 promoter region, a ybbR tag, a Dockerin tag and a T7 terminator. The multilayer microfluidic chip featuring 640 unit cells was aligned to the DNA microarray and bonded to the glass slide. Each unit cell comprised a DNA chamber, a protein chamber, and superseding elastomeric control valves actuated by pneumatic pressure. (b) Control valves were utilized for spatially selective surface modification of each protein chamber with PEG-CoA, and for fluidic isolation of each chamber prior to in vitro expression of the microspotted DNA. Fluorescent labeling with TagRFP-Cohesin was achieved by partial button valve pressurization, leaving only an outer concentric ring of immobilized gene products exposed to the labeling solution. (c) After removal of the microfluidic device, the resulting well-defined, covalently attached protein microarray was accessed from above with a Cohesin-functionalized AFM cantilever. Single-molecule unfolding traces of each of the investigated protein constructs were thus acquired sequentially at each corresponding array address with a single cantilever in a single experiment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4216144&req=5

Figure 1: Method workflow from gene array to single-molecule mechanics.(a) A gene array was spotted onto a glass slide. Genes were designed with a common set of flanking sequences, including a T7 promoter region, a ybbR tag, a Dockerin tag and a T7 terminator. The multilayer microfluidic chip featuring 640 unit cells was aligned to the DNA microarray and bonded to the glass slide. Each unit cell comprised a DNA chamber, a protein chamber, and superseding elastomeric control valves actuated by pneumatic pressure. (b) Control valves were utilized for spatially selective surface modification of each protein chamber with PEG-CoA, and for fluidic isolation of each chamber prior to in vitro expression of the microspotted DNA. Fluorescent labeling with TagRFP-Cohesin was achieved by partial button valve pressurization, leaving only an outer concentric ring of immobilized gene products exposed to the labeling solution. (c) After removal of the microfluidic device, the resulting well-defined, covalently attached protein microarray was accessed from above with a Cohesin-functionalized AFM cantilever. Single-molecule unfolding traces of each of the investigated protein constructs were thus acquired sequentially at each corresponding array address with a single cantilever in a single experiment.
Mentions: Here we developed a platform for parallel characterization of protein mechanics in a single experiment (Fig. 1). Microspotted gene arrays were utilized to synthesize fusion proteins in situ using cell-free gene expression. Proteins were covalently immobilized inside multilayer microfluidic circuits. A single cantilever was then positioned above the protein array, and used to probe the mechanical response of each protein via a common C-terminal Dockerin (Doc) fusion tag. Genes of interest were chosen such that each gene product exhibited an identifiable unfolding pattern when loaded from the N- to C-terminus. Each target protein was expressed with an N-terminal 11 amino acid ybbR tag, which was used to covalently and site-specifically link it to the surface via Sfp Synthase-catalyzed reaction with coenzyme A (CoA)12. At the C-terminus the proteins contained a 75 amino acid cellulosomal Doc from Clostridium thermocellum (C.t.) as specific handle targeted by the Cohesin (Coh)-modified cantilever.

Bottom Line: Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel.A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever.The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

View Article: PubMed Central - PubMed

Affiliation: 1] Lehrstuhl für Angewandte Physik, Ludwig-Maximilians-Universität, Munich, Germany. [2] Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität, Munich, Germany. [3].

ABSTRACT
Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

Show MeSH