Activity control of autodisplayed proteins on the same outer membrane layer of E. coli by using Z-domain/streptavidin/and lipase/foldase systems

Chang, Seo Yoon; Bong, Ji Hong; Yoo, Gu; Lee, Misu; Kang, Min Jung; Jose, Joachim; Pyun, Jae Chul

doi:10.1016/j.enzmictec.2016.10.001

Cited by 7 publications

(3 citation statements)

References 33 publications

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“…For binding confirmation of the screened E. coli to MSU (or CPPD) crystals, plasmids were extracted from the screened E. coli colonies and transformed into E. coli BL21 (DE3) competent cells with the expression vector of eGFP (pKE019), as shown in Figure 2B. 38,39 After induction of Fv antibodies, E. coli (1 mL) at OD 600 nm = 1.0 was mixed with MSU (or CPPD) crystals (2 mg/mL) under mild shaking (10 rpm) for 30 min at 37 °C. Finally, screened E. coli bound to MSU (or CPPD) crystals was observed under a fluorescence microscope (model BX51) from Olympus (Tokyo, Japan), as shown in Figure 2C and 2D.…”

Section: Methodsmentioning

confidence: 99%

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Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout

Jung

Bong

Lee

et al. 2021

ACS Appl. Bio Mater.

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To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (K d) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin–horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0–300 μg/mL.

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Section: Methodsmentioning

confidence: 99%

“…coli colonies and transformed into E. coli BL21 (DE3) competent cells with the expression vector of eGFP (pKE019), as shown in Figure B. , After induction of Fv antibodies, E. coli (1 mL) at OD 600 nm = 1.0 was mixed with MSU (or CPPD) crystals (2 mg/mL) under mild shaking (10 rpm) for 30 min at 37 °C.…”

Section: Experimental Sectionmentioning

confidence: 99%

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout

Jung

Bong

Lee

et al. 2021

ACS Appl. Bio Mater.

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show abstract

“…coli. 20,21 This autodisplay technology has been used for the expression of the Z-domain, 22 streptavidin, 23 lipase, 24 Ro/ SSA antigens, 25 and casein. 26 Using autodisplay technology, the Fv-antibody library was expressed on the outer membrane of E. coli with a high density (>10 5 proteins/E.…”

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confidence: 99%

Screening of Pancreatic Ribonuclease A Inhibitors from an Autodisplayed Fv-Antibody Library

Sung,

Jung,

Hwang

et al. 2023

ACS Pharmacol. Transl. Sci.

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Surface Plasmon Resonance (SPR) Biosensor for the Detection of SARS-CoV-2 Using Autodisplyaed FV-antibodies on Outer Membrane of E. coli

Bong,

Lee,

Jung

et al. 2024

BioChip J

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Activity control of autodisplayed proteins on the same outer membrane layer of E. coli by using Z-domain/streptavidin/and lipase/foldase systems

Cited by 7 publications

References 33 publications

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout

Screening of Pancreatic Ribonuclease A Inhibitors from an Autodisplayed Fv-Antibody Library

Surface Plasmon Resonance (SPR) Biosensor for the Detection of SARS-CoV-2 Using Autodisplyaed FV-antibodies on Outer Membrane of E. coli

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