Autodisplay of the La/SSB protein on LPS-free E. coli for the diagnosis of Sjögren’s syndrome

Yoo, Gu; Dilkaute, Carina; Bong, Ji Hong; Song, Hyun Woo; Lee, Misu; Kang, Min Jung; Jose, Joachim; Pyun, Jae Chul

doi:10.1016/j.enzmictec.2017.01.007

Cited by 9 publications

(7 citation statements)

References 36 publications

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“…In this procedure, induction is performed with an IPTG pulse in the mid-exponential phase (0.6 < OD < 0.8), during a period between 2 and 16 h, with a final IPTG concentration from 0.4 to 1.0 mM. Several studies with ClearColi® BL21(DE3) have used alternative induction strategies (Liang et al 2015 ; Watkins et al 2017b , 2017a ; Yoo et al 2017 ; An et al 2019 ; Hunt et al 2019 ; Sankaran et al 2019 ; Sankaran and del Campo 2019 ; Song et al 2019 ; Wilding et al 2019a , 2019b ; Gnopo et al 2020 ; Segovia-Trinidad et al 2021 ). However, the effects of different induction modes (pulse or auto-induction), inducers, and IPTG and lactose concentrations in recombinant protein production using ClearColi have not previously been investigated in a single study.…”

Section: Discussionmentioning

confidence: 99%

“…To date, most of the published studies with ClearColi® BL21(DE3) have been restricted to complex medium formulations and shake flask cultures to produce enzymes (Liang et al 2015 ; An et al 2019 ; Jobin et al 2019 ; Song et al 2019 ), antigenic proteins (Martínez-Donato et al 2016 ; González-Miro et al 2017 ; Viranaicken et al 2017 ; Watkins et al 2017b , 2017a ; Abdellrazeq et al 2019 , 2020 ), and therapeutic products (Mamat et al 2015 ; Planesse et al 2015 ; Ueda et al 2016 ; Pooe et al 2017 ; Tomczak et al 2017 ; Wang et al 2017 ; Hunt et al 2019 ; Wilding et al 2019a , 2019b ; López et al 2021 ; Nguyen et al 2021 ). ClearColi® BL21(DE3) has also been used to produce diagnostic biomolecules (Masarapu et al 2017 ; Yoo et al 2017 ; Park et al 2021 ) and in specific functional studies (Moon et al 2018 ; Bong et al 2019 ; Hayashi et al 2019 ; Kobayashi et al 2019 ; Qiao et al 2019 ; Carratalá et al 2021 ; Hsu et al 2021 ; Segovia-Trinidad et al 2021 ), among other uses (Ran et al 2019 ; Sankaran et al 2019 ; Sankaran and del Campo 2019 ; Gnopo et al 2020 ). On the other hand, studies have demonstrated that the production of recombinant proteins in conventional E. coli cells is affected by several process factors (Kaur et al 2018 ) that may impact yields and titers, as well as lead to the formation of inclusion bodies and poor protein quality (Rosano and Ceccarelli 2014 ).…”

Section: Introductionmentioning

confidence: 99%

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ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Cardoso

Paredes

Campani

et al. 2022

Appl Microbiol Biotechnol

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Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β- d -thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. Key points • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-11758-9.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

Cardoso

Paredes

Campani

et al. 2022

Appl Microbiol Biotechnol

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“…Bacterial culture conditions for expressing the His6- Sp AS1-autotransporter construct are described in detail in the expanded methods. Additional assessment of correct autodisplay of Sp AS1 was done essentially as described previously. , In short, we expressed the His 6 - Sp AS1-autotransporter construct at 80 mL scale. Half of the cells were treated with proteinase K while the other half was untreated.…”

Section: Methodsmentioning

confidence: 99%

“…Additional assessment of correct autodisplay of SpAS1 was done essentially as described previously. 50,51 In short, we expressed the His 6 -SpAS1-autotransporter construct at 80 mL scale. Half of the cells were treated with proteinase K while the other half was untreated.…”

Section: Construction Of Plasmid Vectors For E Coli Autodisplay Of Smentioning

confidence: 99%

High-Throughput, Lysis-Free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets

et al. 2019

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Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of a high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with E. coli autodisplay. For the first step in a 4-step screening procedure we quantitatively screened >10 5 variants of the homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Display of the sulfatase variants on living E. coli cells ensured the continuous linkage of genotype and phenotype during droplet sorting and allowed for direct recovery by simple regrowth of the sorted cells. The use of autodisplay on living cells simplified and reduced the degree of liquid handling during all steps in the screening procedure to the single event of simply mixing substrate and cells. The percentage of apparent improved variants was enriched >10-fold as a result of droplet sorting. We ultimately identified 25 SpAS1-variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In SpAS1 variants with improved performance toward the bulky fluorescein disulfate, many of the beneficial mutations occur in residues that form hydrogen bonds between α-helices in the C-terminal oligomerization region, suggesting a non-trivial, previously unknown role for the dimer interface in shaping the substrate binding site of SpAS1.

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“…coli by transforming the above-prepared autodisplay vector into competent cells of intact E. coli BL21(DE3), as shown in Figure C. − Transformed E. coli was cultured at 37 °C in Luria–Bertani (LB) broth containing kanamycin (50 mg/L, 10 μL) by shaking (200 rpm) for 16 h. For the main culture, E.…”

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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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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Autodisplay of the La/SSB protein on LPS-free E. coli for the diagnosis of Sjögren’s syndrome

Cited by 9 publications

References 36 publications

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

ClearColi as a platform for untagged pneumococcal surface protein A production: cultivation strategy, bioreactor culture, and purification

High-Throughput, Lysis-Free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets

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

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