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High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae

Paper ID Volume ID Publish Year Pages File Format Full-Text
34471 45027 2015 8 PDF Available
Title
High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae
Abstract

•An integrated process to express and purify WGA A was developed.•Recombinant WGA A was expressed using BEVS in Sf9 cells and R. nu larvae.•An aqueous two-phase system was applied for larval extract clarification.•Chitosan mini-spheres were used to purify WGA A.•Five infected R. nu larvae produced the same amount of WGA A as 1.6 × 108 Sf9 cells.

Wheat germ agglutinin (WGA) is a homodimeric lectin stabilized by non-covalent interactions. Each monomer of 171 residues, which has a complex structure with 16 disulfide bridges, determines two sites for the specific binding of N-acetyl-d-glucosamine and one for the specific binding of N-acetylneuraminic acid. Because of these folding requirements, the production of high yields of recombinant WGA is still not possible and its extraction from wheat germ is the only source for commercial purposes. This work reports for the first time the expression of WGA isolectin A (WGA A), using a baculovirus expression system in Sf9 cells and Rachiplusia nu larvae. High levels of recombinant WGA A were obtained in both cases, especially in R. nu, where yields reached 346.6 ± 88.5 μg/g of larvae. Also, an integrated purification method was developed based on aqueous two-phase separation coupled to affinity chromatography using chitosan mini-spheres. The recombinant WGA A was able to recognize ovoalbumin sugar moieties, cross-react with anti-WGA serum and agglutinate human red blood cells, and showed the same behavior as that of commercial WGA in SDS-PAGE and RP-HPLC analyses.

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Keywords
Wheat germ agglutinin; Baculovirus; Larvae; Aqueous two-phase system; Chitosan mini-spheres
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High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 1, January 2015, Pages 40–47
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us