Construction of a Fusion Enzyme System by Gene Splicing as a New Molecular Recognition Element for a Sequence Biosensor

Zhou, Ya‐Feng; Zhang, Xian-En; Liu, Hong; Zhang, Zhiping; Zhang, Chenggang; Cass, Anthony E. G.

doi:10.1021/bc015509d

Cited by 19 publications

(8 citation statements)

References 30 publications

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“…The linker peptide (repeat serine and glycin sequence) is a rigid and hydrophilic a-helices chain in solution (Shi et al 2004), which plays a spacer to help to correct folding of each fusion partners. The linker peptide function had been demonstrated in our previous studies and many other investigations (Freund et al 1993;Zhou et al 2001;Shao et al 2001) and worked well again in this study. …”

Section: Binding Characteristics Of Scfv and Scfv Fusion Pair Throughsupporting

confidence: 88%

Construction and Characterization of an Anti-Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Zhang¹,

Zhang²,

Zhou³

et al. 2007

LANL

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A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin-mature bovine prion fusion protein (TrxA-bPrP c ). After five rounds of panning against recombinant bovine prion protein (rb-PrP c ) and ELISA test, two positive clones with high affinity to rb-PrP c , named Z163 and Z186, were obtained. They were conjugated with a linker-streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186-L-SBP/Z163-Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27-30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb-PrP c were 3.24 Â 10 28 M, 8.82 Â 10 28 M, and 8.10 Â 10 29 M for Z186-L-SBP, Z163, and Z163-Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186-L-SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163-Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb-PrP c with detection limit of 1 pg/ml.

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Section: Binding Characteristics Of Scfv and Scfv Fusion Pair Throughsupporting

confidence: 88%

Construction and Characterization of an Anti-Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Zhang¹,

Zhang²,

Zhou³

et al. 2007

LANL

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“…The addition of the linker peptides provides spacers to minimize steric hindrance among the functional components of the fusion protein. 30 This fusion molecular system, THLSLM, could be efficiently immobilized on a streptavidin-modified chip with controlled homogeneity through streptavidin-Strep-tag II interaction.…”

Section: Resultsmentioning

confidence: 99%

“…The method proposed here describes the attempt of using the fusion molecular system THLSLM, constructed by gene splicing in vitro, to make a protein chip for specific detection of mutations. The addition of the linker peptides provides spacers to minimize steric hindrance among the functional components of the fusion protein . This fusion molecular system, THLSLM, could be efficiently immobilized on a streptavidin-modified chip with controlled homogeneity through streptavidin-Strep-tag II interaction.…”

Section: Resultsmentioning

confidence: 99%

A MutS-Based Protein Chip for Detection of DNA Mutations

Zhou

Zhang

et al. 2003

Anal. Chem.

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This paper describes a new protein chip method for detection of single-base mismatches and unpaired bases of DNA, using a genetic fusion molecular system Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutS (THLSLM). The THLSLM coding sequence was constructed by attaching Strep-tag II and mutS gene to pET32a (+) sequentially with insertion of a linker peptide coding sequence before and behind Strep-tagII gene, respectively. THLSLM was expressed in E. coli AD494 (DE3) and purified using Ni(2+)-chelation affinity resin. THLSLM retained both mismatch recognition activity and streptavidin binding affinity. THLSLM was then immobilized on the chip matrix coated with streptavidin through the Strep-tag II-streptavidin binding reaction. The resulting protein chip was used to detect the mismatched and unpaired mutations in the synthesized oligonucleotides, as well as a single-base mutation in rpoB gene from Mycobacterium tuberculosis, with high specificity. The method could potentially serve as a platform to develop the high-throughput technology for screening and analysis of genetic mutations.

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“…In addition to improved stability and sensitivity, a convenient “tag” could be employed as a fusion partner for ease of detection. A bifunctional fusion enzyme system constructed for a maltose biosensor was developed by gene splicing technique [148]. The fusion enzyme system of glucoamylase (GA, (E.C 3.2.1.3)) and GOx, by fusing the complementary DNA (cDNA) fragment of Aspergillus niger glucoamylase to the 3’ end of the A. niger GOx gene with the insertion of a flexible linker peptide (-(Ser-Gly)5-) coding sequence was reported by Chen et al [149].…”

Section: Approaches In Improving Enzyme Usage In Biosensorsmentioning

confidence: 99%

Immobilized Enzymes in Biosensor Applications

et al. 2019

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Enzyme-based biosensing devices have been extensively developed over the last few decades, and have proven to be innovative techniques in the qualitative and quantitative analysis of a variety of target substrates over a wide range of applications. Distinct advantages that enzyme-based biosensors provide, such as high sensitivity and specificity, portability, cost-effectiveness, and the possibilities for miniaturization and point-of-care diagnostic testing make them more and more attractive for research focused on clinical analysis, food safety control, or disease monitoring purposes. Therefore, this review article investigates the operating principle of enzymatic biosensors utilizing electrochemical, optical, thermistor, and piezoelectric measurement techniques and their applications in the literature, as well as approaches in improving the use of enzymes for biosensors.

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Construction of a Fusion Enzyme System by Gene Splicing as a New Molecular Recognition Element for a Sequence Biosensor

Cited by 19 publications

References 30 publications

Construction and Characterization of an Anti-Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Construction and Characterization of an Anti-Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

A MutS-Based Protein Chip for Detection of DNA Mutations

Immobilized Enzymes in Biosensor Applications

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