Enhancement of solubility and yield of a β-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag

Dulal, Hari Prasad; Nagae, Masamichi; Ikeda, Akemi; Morita-Matsumoto, Kana; Adachi, Yoshiyuki; Ohno, Naohito; Yamaguchi, Yoshiki

doi:10.1016/j.pep.2016.04.002

Cited by 17 publications

(13 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Seamless cloning methods generally rely on short (~15 bp) end homology regions for ligation of insert and vector DNA fragments. These methods are available through commercial kits, which are widely used [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] ; however, seamless cloning kits are cost-prohibitive. Recently, several simple and recombinant enzyme-free seamless DNA cloning methods have been described [15] , [16] , [17] , [18] , which utilize the endogenous homologous recombination activity of laboratory Escherichia coli strains.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Motohashi

2017

Biochemistry and Biophysics Reports

View full text Add to dashboard Cite

Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC) can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE) cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp) end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid engineering.

show abstract

Section: Introductionmentioning

confidence: 99%

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Motohashi

2017

Biochemistry and Biophysics Reports

View full text Add to dashboard Cite

show abstract

“…A wide variety of protein chemical manipulations have been applied to improve sDectin-1 solubility with mixed success. For example, the solubility and utility of sDectin-1 was increased by tethering it to the 56 amino acid long B1 domain of Streptococcal Protein G (45) or more commonly to the 232 a.a. long Fc constant region of IgG1 antibody (41). Bacterially produced non-glycosylated sDectin-1 renatured from inclusion bodies and de-glycosylated and native mammalian cell produced sDectin-1 all retain indistinguishable beta-glucan binding activity (39, 46, 47).…”

Section: Discussionmentioning

confidence: 99%

“…S2 ). Samples of sDectin-1 at 6 ug/uL in this same GuHCl buffer were adjusted to pH 8.3 with 1 M pH =10 triethanolamine and reacted with a 4-molar excess of DSPE-PEG-3400-NHS (Nanosoft polymers, 1544-3400) for 1 hr at 23°C to make DSPE-PEG-DEC. Gel exclusion chromatography on Bio-Gel P-6 acrylamide resin (Bio-Rad #150-0740) in renaturation and storage buffer RN#5 (0.1 M NaH2PO4, 10 mM Triethanolamine, pH 8.0, 1 M L-Arginine, 100 mM NaCl, 5 mM EDTA, 5 mM BME) removed un-incorporated DSPE-PEG and GuHCl (45, 57). DSPE-PEG-BSA was prepared from bovine serum albumin BSA (Sigma, A-8022) by the same protocol, minus the GuHCl from DSPE-PEG labeling buffers and L-Arginine from RN#5 buffer.…”

Section: Methodsmentioning

confidence: 99%

Targeted antifungal liposomes

Ambati

Ferarro

Khang

et al. 2019

Preprint

View full text Add to dashboard Cite

15 Aspergillus species cause pulmonary invasive aspergillosis resulting in nearly a hundred 16 thousand deaths each year. Patients at the greatest risk of developing life-threatening 17 aspergillosis have weakened immune systems and/or various lung disorders. Patients are 18 treated with antifungals such as amphotericin B (AmB), casofungin acetate, or triazoles 19 (itraconazole, voriconazole etc.), but these antifungal agents have serious limitations due to lack 20 of sufficient fungicidal effect and human toxicity. Liposomes with AmB intercalated into the lipid 21 membrane (AmBisomes, AmB-LLs), have several-fold reduced toxicity compared to detergent 22 Importance 41 The fungus Aspergillus fumigatus causes pulmonary invasive aspergillosis resulting in 42 nearly a hundred thousand deaths each year. Patients are often treated with antifungal drugs 43 such as amphotericin B loaded into liposomes, AmB-LLs, but all antifungal drugs including 44 AmB-LLs have serious limitations due to human toxicity and insufficient fungal cell killing. Even 45with the best current therapies, one-year survival among patients with invasive aspergillosis is 46 only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. 47Dectin-1 is a mammalian protein that binds to beta-glucan polysaccharides found in 48 nearly all fungal cell walls. We coated AmB-LLs with Dectin-1 to make DEC-AmB-LLs. DEC-49

show abstract

“…On the other hand, glycans can modulate the functions of immunoglobulins (IgGs), which exhibit unique properties depending on how they are glycosylated . The antibody‐dependent cellular cytotoxicity (ADCC) of IgG antibodies, indeed, is reduced by the presence of fucose on the glycan core …”

Section: Figurementioning

confidence: 99%

“…Furthermore, modified IgG Fc glycosylation impacts antibody conformation and influences its capacity to interact with immune Fc receptors and complement proteins. It should be noted that ADCC is strongly reduced by the presence of fucose on the pentasaccharide core of N ‐glycan in the Fc region of IgG antibodies . On the other hand, core fucosylation is required for the appropriate activation of different signaling pathways, including the release of inflammatory cytokines TGF‐β1 and the epidermal and vascular endothelial growth factors…”

Section: Figurementioning

confidence: 99%

The Core Fucose on an IgG Antibody is an Endogenous Ligand of Dectin‐1

et al. 2019

Self Cite

View full text Add to dashboard Cite

The core fucose,amajor modification of N-glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via the promotion of PD-1 expression on Tc ells.A lthough the core fucose regulates many biological processes,n oc ore fucose recognition molecule has been identified in mammals.Herein, we report that Dectin-1, ak nowna nti-b-glucan lectin, recognizes the core fucose on IgG antibodies.Acombination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N-terminal asparagine at the glycosylation site as well as the core fucose. Thus,D ectin-1 appears to be the first lectin-like molecule involved in the heterovalent and specific recognition of characteristic N-glycans on antibodies.

show abstract

Enhancement of solubility and yield of a β-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag

Cited by 17 publications

References 32 publications

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Targeted antifungal liposomes

The Core Fucose on an IgG Antibody is an Endogenous Ligand of Dectin‐1

Contact Info

Product

Resources

About