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A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Bottom Line: We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames.Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles.As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.

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Expression and characterization of 13FNIII and VWFA2 tandem repeat proteins.Superposed elution profiles from size exclusion chromatography of (13FNIII)2–8 proteins (A) and (VWFA2)6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)n unfolding curves in the presence and absence of Ca2+.
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pone-0037617-g004: Expression and characterization of 13FNIII and VWFA2 tandem repeat proteins.Superposed elution profiles from size exclusion chromatography of (13FNIII)2–8 proteins (A) and (VWFA2)6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)n unfolding curves in the presence and absence of Ca2+.

Mentions: To verify our assembly format, constructs (13FNIII)2,4,6,8 and (A2)6,8,10 were expressed in HEK293-EBNA1 (HEK-E) cells and purified by immobilized metal affinity chromatography followed by size exclusion chromatography (Fig. 4A-D). We performed thermal denaturation assays to assess the folding state of the purified proteins and to investigate the effect of repeat number on thermal stability. All constructs display a two-state unfolding transition from a stable fluorescence baseline (Fig. 4E,F). We observed a gradual decrease in melting temperature (Tm) with increasing number of tandem repeats for the concatameric (13FNIII)n constructs (Table 2). Consistently, the unfolding temperature of 62.3±0.2°C for the dimeric 13FNIII construct is lower than the value of 72°C reported for the monomer [21]. In contrast, the series of concatameric VWFA2 constructs showed little variation in thermal stability. We have previously reported on the modulation of thermodynamic and mechanical stability of VWFA2 by binding of Ca2+ to a highly conserved calcium-binding site [18]. In accordance, we find a marked Tm shift for buffer conditions containing either Ca2+ or EDTA for the tandem domain constructs, indicating that the VWFA2 domains fold into their native structure also in this repetitive context.


A rapid cloning method employing orthogonal end protection.

Jakobi AJ, Huizinga EG - PLoS ONE (2012)

Expression and characterization of 13FNIII and VWFA2 tandem repeat proteins.Superposed elution profiles from size exclusion chromatography of (13FNIII)2–8 proteins (A) and (VWFA2)6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)n unfolding curves in the presence and absence of Ca2+.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369885&req=5

pone-0037617-g004: Expression and characterization of 13FNIII and VWFA2 tandem repeat proteins.Superposed elution profiles from size exclusion chromatography of (13FNIII)2–8 proteins (A) and (VWFA2)6–10 (B). (C,D) Coomassie stained SDS-PAGE of the purified proteins. (E,F) Unfolding curves from Thermofluor analysis suggest that the concatameric constructs are properly folded. Note the consistent shift of the (VWFA2)n unfolding curves in the presence and absence of Ca2+.
Mentions: To verify our assembly format, constructs (13FNIII)2,4,6,8 and (A2)6,8,10 were expressed in HEK293-EBNA1 (HEK-E) cells and purified by immobilized metal affinity chromatography followed by size exclusion chromatography (Fig. 4A-D). We performed thermal denaturation assays to assess the folding state of the purified proteins and to investigate the effect of repeat number on thermal stability. All constructs display a two-state unfolding transition from a stable fluorescence baseline (Fig. 4E,F). We observed a gradual decrease in melting temperature (Tm) with increasing number of tandem repeats for the concatameric (13FNIII)n constructs (Table 2). Consistently, the unfolding temperature of 62.3±0.2°C for the dimeric 13FNIII construct is lower than the value of 72°C reported for the monomer [21]. In contrast, the series of concatameric VWFA2 constructs showed little variation in thermal stability. We have previously reported on the modulation of thermodynamic and mechanical stability of VWFA2 by binding of Ca2+ to a highly conserved calcium-binding site [18]. In accordance, we find a marked Tm shift for buffer conditions containing either Ca2+ or EDTA for the tandem domain constructs, indicating that the VWFA2 domains fold into their native structure also in this repetitive context.

Bottom Line: We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames.Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles.As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs.

View Article: PubMed Central - PubMed

Affiliation: Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, The Netherlands.

ABSTRACT
We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.

Show MeSH
Related in: MedlinePlus