Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli

Yang, Jong Min; Kim, Gi Eob; Kim, Kyeong Rok; Kim, Chang Sup

doi:10.1016/j.enzmictec.2019.109489

Cited by 3 publications

(3 citation statements)

References 33 publications

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“…[30] On the other hand, CjCgtBΔ30-His 6 with an expression level of 20 mg purified protein per liter culture [31] was more stable but its expression level had room for improvement. To increase their soluble expression levels and stability, [32] an maltosebinding protein (MBP) was fused to the N-terminus of Δ15CjCgtA-His 6 and CjCgtBΔ30-His 6 . The expression levels of the resulting recombinant MBP-Δ15CjCgtA-His 6 (Figure S1, Supporting Information) and MBP-CjCgtBΔ30-His 6 (Figure S2, Supporting Information) were improved to 85 mg L À 1 culture and 110 mg L À 1 culture, respectively (Figure S3, Supporting Information).…”

Section: Glycosyltransferase Improvement and Characterizationmentioning

confidence: 99%

Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides

Zhang

Yang

et al. 2023

Chemistry A European J

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Large-scale synthesis of GM1, an important ganglioside in mammalian cells especially those in the nervous system, is needed to explore its therapeutic potential. Biocatalytic production is a promising platform for such a purpose. We report herein the development of process engineering and glycosyltransferase improvement strategies to advance chemoenzymatic total synthesis of GM1. Firstly, a new short route was developed for chemical synthesis of lactosylsphingosine from the commercially available Garner's aldehyde. Secondly, two glycosyltransferases including Campylobacter jejuni β1-4GalNAcT (CjCgtA) and β1-3-galactosyltransferase (CjCgtB) were improved on their soluble expression in E. coli and enzyme stability by fusing with an N-terminal maltose binding protein (MBP). Thirdly, the process for enzymatic synthesis of GM1 sphingosines from lactosylsphingosine was engineered by developing a multistep onepot multienzyme (MSOPME) strategy without isolating intermediate glycosphingosines and by adding a detergent, sodium cholate, to the later enzymatic glycosylation steps. Installation of a desired fatty acyl chain to GM1 glycosphingosines led to the formation of target GM1 gangliosides. The combination of glycosyltransferase improvement with chemical and enzymatic process engineering represents a significant advance in obtaining GM1 gangliosides containing different sialic acid forms by total chemoenzymatic synthesis in a short route and with high efficiency.

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Section: Glycosyltransferase Improvement and Characterizationmentioning

confidence: 99%

Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides

Zhang

Yang

et al. 2023

Chemistry A European J

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“…12 In this chemoenzymatic method, β-(1,4)-N-acetylgalactosaminyltransferase was originally from Campylobacter jejuni (CgtA), which is responsible for the extension of Neu5Acα and GM2 gangliosides. 13 Wen et al found CgtA could also tolerate unnatural GalNAz (an azide group at C-2 of natural GalNAc), forming the Neu5Acα2−3(GalNAzβ1−4)Gal trisaccharide. Then, the introduced azide group will react with alkyne fluorescence via click chemistry, e.g., copper-catalyzed azide−alkyne cycloaddition (CuAAC) and copper-free strain promoted alkyne−azide cycloaddition (SPACC).…”

mentioning

confidence: 99%

“…In this chemoenzymatic method, β-(1,4)-N-acetylgalactosaminyltransferase was originally from Campylobacter jejuni (CgtA), which is responsible for the extension of Neu5Acα(2–3)Gal with the GalNAc residue forming GM1 and GM2 gangliosides . Wen et al found CgtA could also tolerate unnatural GalNAz (an azide group at C-2 of natural GalNAc), forming the Neu5Acα2–3(GalNAzβ1–4)Gal trisaccharide.…”

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confidence: 99%

Chemoenzymatic Labeling Pathogens Containing Terminal N-Acetylneuraminic Acid−α(2–3)-Galactose Glycans

Chao

et al. 2022

ACS Infect. Dis.

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The N-acetylneuraminic acid−α(2–3)-galactose epitope is often located at the nonreducing terminal ends of glycans on the envelopes of many pathogens, and it is believed that this structure mimics a host’s oligosaccharide so as to circumvent and/or counteract the host’s immune responses. A chemoenzymatic method for the rapid and sensitive detection of N-acetylneuraminic acid−α(2–3)-galactose has been built, so we planned to examine whether the chemoenzymatic method could be applied on the detection of N-acetylneuraminic acid−α(2–3)-galactose on pathogens. Our results revealed that the chemoenzymatic method was rapid and sensitive for labeling live or dead Gram-positive Streptococcus agalactiae A909 and Gram-negative Campylobacter jejuni MK104 with N-acetylneuraminic acid−α(2–3)-galactose. This study suggested that the chemoenzymatic method was a new strategy for labeling pathogens and had potential for the diagnosis of or therapeutics for pathogenic infection.

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Utilization of glycosyltransferases as a seamless tool for synthesis and modification of the oligosaccharides-A review

Ali

Liaqat

Khazi

et al. 2023

International Journal of Biological Macromolecules

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Expression and purification of the full-length N-acetylgalactosaminyltransferase and galactosyltransferase from Campylobacter jejuni in Escherichia coli

Cited by 3 publications

References 33 publications

Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides

Process Engineering and Glycosyltransferase Improvement for Short Route Chemoenzymatic Total Synthesis of GM1 Gangliosides

Chemoenzymatic Labeling Pathogens Containing Terminal N-Acetylneuraminic Acid−α(2–3)-Galactose Glycans

Utilization of glycosyltransferases as a seamless tool for synthesis and modification of the oligosaccharides-A review

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