Linkers: a synergistic way for chimeric proteins

Patel, Dharti Keyur; Menon, Dhanya V.; Patel, Darshan H.

doi:10.22541/au.160199719.94542802/v1

Cited by 2 publications

(2 citation statements)

References 54 publications

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“…Tethering of catalytic and CBM domains provides distinct advantages in polysaccharide degradation: increase effective enzyme concentrations on the polysaccharide surface; target of catalytic module to the substrate; and disruption of the polysaccharide structure [ 21 ]. A two-domain construct, GH11-CBM36-1, previously cloned and biochemically characterised [ 14 ] was structurally analysed in this study.…”

Section: Resultsmentioning

confidence: 99%

Structural and biophysical characterization of the multidomain xylanase Xyl

2022

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The depletion of fossil fuels, associated pollution, and resulting health hazards are of concern worldwide. Woody biomass constitutes an alternative source of cleaner and renewable energy. The efficient use of woody biomass depends on xylan depolymerisation as the endo-β-1,4-xylopyranosyl homopolymer is the main component of hemicellulose, the second most abundant component of wood. Xylan depolymerisation is achieved by hemicellulolytic xylanases of glycoside hydrolase (GH) families 5, 8, 10, 11, 30 and 43 of the CAZY database. We analysed a multidomain xylanase (Xyl) from the hindgut metagenome of the snouted harvester termite Trinervitermes trinervoides that releases xylobiose and xylotriose from beech and birch xylan and wheat arabinoxylan. The four domains of Xyl include an N-terminal GH11 xylanase domain, two family 36-like carbohydrate-binding domains CBM36-1 and 2, and a C-terminal CE4 esterase domain. Previous analyses indicated that CBM36-1 deletion slightly increased GH11 catalysis at low pH whereas removal of both CBMs decreased xylanase activity at 60°C from 90 to 56%. Possible cooperativity between the domains suggested by these observations was explored. A crystal structure of the two-domain construct, GH11-CBM36-1, confirmed the structure of the GH11 domain whereas the CBM36-1 domain lacked electron density, possibly indicating a random orientation of the CBM36-1 domain around the GH11 domain. Isothermal titration calorimetry (ITC) experiments similarly did not indicate specific interactions between the individual domains of Xyl supporting a “beads-on-a-string” model for Xyl domains.

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Section: Resultsmentioning

confidence: 99%

Structural and biophysical characterization of the multidomain xylanase Xyl

2022

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“…On their structure, linkers are divided into three categories, namely flexible, rigid, and in vivo cleavable. Linkers maintain an appropriate distance between the peptides so that each has its function ( 36 ). Low protein production, impaired biological activity, and proteins misfolding are the consequences of using an inappropriate linker or not using a linker ( 37 ).…”

Section: Discussionmentioning

confidence: 99%

In Silico Vaccine Design and Expression of the Multi-Component Protein Candidate against the Toxoplasma gondii Parasite from MIC13, GRA1, and SAG1 Antigens

Hosseininejad,

Daryani,

Fasihi-Ramandi

et al. 2023

IJPA

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Background: We aimed to design a B and T cell recombinant protein vaccine of Toxoplasma gondii with in silico approach. MIC13 plays an important role in spreading the parasite in the host body. GRA1 causes the persistence of the parasite in the parasitophorous vacuole. SAG1 plays a role in host-cell adhesion and cell invasion. Methods: Amino acid positions 73-272 from MIC13, 71-190 from GRA1, and 101-300 from SAG1 were selected and joined with linker A(EAAAK)A. The structures, antigenicity, allergenicity, physicochemical properties, as well as codon optimization and mRNA structure of this recombinant protein called MGS1, were predicted using bioinformatics servers. The designed structure was synthesized and then cloned in pET28a (+) plasmid and transformed into Escherichia coli BL21. Results: The number of amino acids in this antigen was 555, and its antigenicity was estimated to be 0.6340. SDS-PAGE and Western blotting confirmed gene expression and successful production of the protein with a molecular weight of 59.56kDa. This protein will be used in our future studies as an anti-Toxoplasma vaccine candidate in animal models Conclusion: In silico methods are efficient for understanding information about proteins, selecting immunogenic epitopes, and finally producing recombinant proteins, as well as reducing the time and cost of vaccine design.

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Linkers: a synergistic way for chimeric proteins

Cited by 2 publications

References 54 publications

Structural and biophysical characterization of the multidomain xylanase Xyl

Structural and biophysical characterization of the multidomain xylanase Xyl

In Silico Vaccine Design and Expression of the Multi-Component Protein Candidate against the Toxoplasma gondii Parasite from MIC13, GRA1, and SAG1 Antigens

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