Bglbrick strategy for the construction of single domain antibody fusions

Goldman, Ellen R.; Broussard, Aeris; Anderson, George P.; Liu, Jinny L.

doi:10.1016/j.heliyon.2017.e00474

Cited by 5 publications

(8 citation statements)

References 24 publications

(47 reference statements)

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“…enzyme–Hoc) fusions with a 6 × His tag by co-expressing enzyme–SC and St–Hoc in the same E. coli host. The fusion was purified via Ni-Sepharose resins, and separated from un-fused enzyme–SC through size exclusion chromatography (Goldman et al, 2017 ). Larger enzyme–Hoc fusions eluted earlier than smaller enzyme–SC fusions from the size-exclusion column ( Figure S2 ).…”

Section: Resultsmentioning

confidence: 99%

“…All the primer sequences used for cloning are listed in Table S1 and gene maps and sequences are described in the Supplementary Information . The enzyme–SC fusions were cloned into pET28, while St–Hoc was inserted into pACYCduet according to Anderson et al ( 2010 ) and Goldman et al ( 2017 ). Details are provided in the Supplementary Information .…”

Section: Methodsmentioning

confidence: 99%

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Multi-Enzyme Assembly on T4 Phage Scaffold

Liu

Zabetakis

Breger

et al. 2020

Front. Bioeng. Biotechnol.

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Over the past two decades, various scaffolds have been designed and synthesized to organize enzyme cascades spatially for enhanced enzyme activity based on the concepts of substrate channeling and enhanced stability. The most bio-compatible synthetic scaffolds known for enzyme immobilization are protein and DNA nanostructures. Herein, we examined the utility of the T4 phage capsid to serve as a naturally occurring protein scaffold for the immobilization of a three-enzyme cascade: Amylase, Maltase, and Glucokinase. Covalent constructs between each of the enzymes and the outer capsid protein Hoc were prepared through SpyTag-SpyCatcher pairing and assembled onto phage capsids in vitro with an estimated average of 90 copies per capsid. The capsid-immobilized Maltase has a fourfold higher initial rate relative to Maltase free in solution. Kinetic analysis also revealed that the immobilized three-enzyme cascade has an 18-fold higher converted number of NAD + to NADH relative to the mixtures in solution. Our results demonstrate that the T4 phage capsid can act as a naturally occurring scaffold with substantial potential to enhance enzyme activity by spatially organizing enzymes on the capsid Hoc.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Multi-Enzyme Assembly on T4 Phage Scaffold

Liu

Zabetakis

Breger

et al. 2020

Front. Bioeng. Biotechnol.

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“…To achieve this goal, the two best options are to immunize another animal in an effort to obtain an immune repertoire that possesses higher affinity binders or to take advantage of avidity by preparing sdAb multimers of our current binders. In other work we, as well as others, have made sdAb multimers either via direct genetic linkage [ 29 , 30 , 31 ] or via a fusion protein that forms a homodimer such as alkaline phosphatase or rhizavidin [ 32 , 33 ] or fusion with a peptide that promotes multimerization [ 34 ]. These dimers and multimers have been shown to dramatically enhance both affinity and utility in immunoassays [ 35 , 36 ], however, it remains to be determined if such an approach would prove successful here.…”

Section: Discussionmentioning

confidence: 99%

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein

et al. 2020

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Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents.

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“…The Bgl II site was removed from the pET22b vector using the quick change mutagenesis kit (Agilent), and a Bglbrick compatible version of pET22b termed pET22b- Bgl II was constructed. 27 The DNA coding for each of the SYNZIP sequences 12 was synthesized by Eurofins Genomics, with PCR used to introduce linkers and restriction sites for Bglbrick cloning. 18 Each sdAb was also cloned into pET22b- Bgl II.…”

Section: Experimental Sectionmentioning

confidence: 99%

Orthogonal Synthetic Zippers as Protein Scaffolds

et al. 2018

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Protein scaffolds have proven useful for co-localization of enzymes, providing control over stoichiometry and leading to higher local enzyme concentrations, which have led to improved product formation. To broaden their usefulness, it is necessary to have a wide choice of building blocks to mix and match for scaffold generation. Ideally, the scaffold building blocks should function at any location within the scaffold and have high affinity interactions with their binding partners. We examined the utility of orthogonal synthetic coiled coils (zippers) as scaffold components. The orthogonal zippers are coiled coil domains that form heterodimers only with their specific partner and not with other zipper domains. Focusing on two orthogonal zipper pairs, we demonstrated that they are able to function on either end or in the middle of a multiblock assembly. Surface plasmon resonance was employed to assess the binding kinetics of zipper pairs placed at the start, middle, or end of a construct. Size-exclusion chromatography was used to demonstrate the ability of a scaffold with two zipper domains to bind their partners simultaneously. We then expanded the study to examine the binding kinetics and cross-reactivities of three additional zipper pairs. By validating the affinities and specificities of synthetic zipper pairs, we demonstrated the potential for zipper domains to provide an expanded library of scaffolding parts for tethering enzymes in complex pathways for synthetic biology applications.

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Bglbrick strategy for the construction of single domain antibody fusions

Cited by 5 publications

References 24 publications

Multi-Enzyme Assembly on T4 Phage Scaffold

Multi-Enzyme Assembly on T4 Phage Scaffold

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein

Orthogonal Synthetic Zippers as Protein Scaffolds

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