Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

doi:10.1016/j.jbiosc.2015.06.003

Cited by 10 publications

(6 citation statements)

References 35 publications

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“…Moreover, the binding affinity ratio between the 17‐bp ORC and nonspecific DNA with scCro is 10,000:1, which indicates a low probability of interference with any other sequence than the ORC (Kim, Takeda, Matthews, & Anderson, 1987). Leveraging the high affinity and specificity, scCro has been successfully utilized for protein immobilization in microbead display (Kojima et al, 2016; Zhu et al, 2015) and to control the spatial arrangement of enzymes on DNA scaffolds for designing reaction cascades (Kojima et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins

Zhu

Gan

Cabezas

et al. 2020

Biotech & Bioengineering

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In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates. K E Y W O R D S cell-free protein synthesis, CroP-LET, in vitro transcription/translation, linear expression template, scCro, Vibrio natriegens Abbreviations: CFPS, cell-free protein synthesis; LET, linear expression template; scCro, single-chain derivative of the bacteriophage lambda Cro repressor; sfGFP, superfolder green fluorescent protein. Bo Zhu and Rui Gan contributed equally to this work.

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Section: Introductionmentioning

confidence: 99%

Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins

Zhu

Gan

Cabezas

et al. 2020

Biotech & Bioengineering

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“…Commercially available beads made of polystyrene (PS), silica (SiO 2 ), or poly(methylmethacrylate) (PMMA) are currently used for detection of analytes and for multiplexing studies that are read out by flow cytometry [ 1 , 25 ]. We wanted to directly compare these beads with polyelectrolyte microcapsules with respect to their binding capacity for antibodies and their sensitivity for protein and nucleic acid analytes.…”

Section: Resultsmentioning

confidence: 99%

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids

et al. 2018

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To detect and study diseases, research and clinical laboratories must quantify specific biomarkers in the plasma and urine of patients with precision, sensitivity, and cost-effectiveness. Newly developed techniques, such as particle-based immunoassays, must be validated in these terms against standard methods such as enzyme-linked immunosorbent assays (ELISAs). Here, we compare the performance of assays that use hollow polyelectrolyte microcapsules with assays based on solid plastic beads, and with standard microplate immunoassays. The polyelectrolyte microcapsules detect the disease biomarker beta-2 microglobulin with a fifty-fold increase in sensitivity than polystyrene (PS) beads. For sequence-specific nucleic acid detection, the oligonucleotide-coated microcapsules exhibit a two-fold lower increase in sensitivity over PS beads. The microcapsules also detect the presence of a monoclonal antibody in hybridoma supernatant at a fifty-six-fold increase in sensitivity compared to a microplate assay. Overall, polyelectrolyte microcapsule-based assays are more sensitive for the detection of protein and nucleic acid analytes than PS beads and microplate assays, and they are viable alternatives as a platform for the rapid quantitative detection of analytes at very low concentrations.

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“…The scCro expression plasmid pET22-scCro previously described (Kojima et al, 2016) was transformed into the E. coli strain BL21(DE3). A 100 mL of 2 × YT medium containing 50 µg/mL carbenicillin was inoculated with 1 mL of overnight pre-culture.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, the binding affinity ratio between the 17-bp ORC and non-specific DNA with scCro is 10,000:1, which indicates a low probability of interference with any other sequence than the ORC (Kim et al, 1987). Leveraging the high affinity and specificity, scCro has been successfully utilized for protein immobilization in microbead display (Kojima et al, 2016; Zhu et al, 2015) and to control the spatial arrangement of enzymes on DNA scaffolds for designing reaction cascades (Kojima et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Increasing cell-free gene expression yields from linear templates inEscherichia coliandVibrio natriegensextracts by using DNA-binding proteins

Zhu

Gan

Cabezas³

et al. 2020

Preprint

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In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based Protection of LET) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

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Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays

Cited by 10 publications

References 35 publications

Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins

Increasing cell‐free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA‐binding proteins

Comparative validation of a microcapsule-based immunoassay for the detection of proteins and nucleic acids

Increasing cell-free gene expression yields from linear templates inEscherichia coliandVibrio natriegensextracts by using DNA-binding proteins

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