Lentil lectin affinity chromatography

Manufactured by GE Healthcare

Sourced in United States

Lentil-lectin affinity chromatography is a technique used to purify and isolate specific proteins from complex mixtures. It utilizes the reversible binding interactions between lectins, which are carbohydrate-binding proteins, and their corresponding glycosylated target proteins. This method allows for the selective capture and recovery of the desired proteins while removing unwanted components.

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Lab products found in correlation

Freestyle 293 f suspension cells, by Thermo Fisher Scientific (2 mentions) Nickel chelating chromatography, by GE Healthcare (1 mentions) Bira biotin ligase, by Avidity (1 mentions)

3 protocols using lentil lectin affinity chromatography

Transient Transfection and Purification of Recombinant gp120-BC Protein

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The recombinant gp120-BC protein was produced by transient transfection of FreeStyle™ 293F suspension cells (Invitrogen, Carlsbad, CA) [9 (link)] using the same gp120-encoding DNA vaccine plasmid. In brief, cells were transfected at a density of 1 × 10⁶/ml in GIBCO® FreeStyle™ 293 expression media using 293fectin™, according to manufacturer’s instructions (Invitrogen). Three days after the transfection, supernatant was collected and the gp120 protein was purified by lentil-lectin affinity chromatography (GE Healthcare, Chicago, IL). The purified gp120-BC protein was verified by ELISA and Western-blot analysis.

Li H., Wang S., Hu G., Zhang L., Liu S, & Lu S. (2021). DNA priming immunization is more effective than recombinant protein vaccine in eliciting antigen-specific B cell responses. Emerging Microbes & Infections, 10(1), 833-841.

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Production and Purification of HIV-1 Env Proteins

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The soluble YU2-derived gp140-F trimers (36 (link)) used as the immunogen were produced by transient transfection into Freestyle 293F suspension cells (Invitrogen) as previously described (37 (link)). The Env ligands used in the binding studies were the following trimeric proteins: gp140-F, gp120-F, gp120-F-ΔV3, gp120-F-ΔV1V2, gp140-F-D368R, and the following monomeric proteins: TriMut, TriMut-368/370, TriMut-368/370/474 and gp140-GCN4 were purified by lentil-lectin and gel filtration chromatography. The biotinylated gp140-F probe used for single cell sorting by flow cytometry was purified by lentil-lectin affinity chromatography and nickel-chelating chromatography (GE Healthcare, Uppsala, Sweden). All probes carried an Avi-tag for site-specific biotinylation at the C-termini of the proteins and biotinylation was performed with biotin ligase Bir A (Avidity, Denver, CO). All Env proteins were from the YU2 strain except the TriMut proteins, which were from HXBc2. The collagen-foldon protein was kindly received from the laboratory of Professor Rikard Holmdahl at Karolinska Institutet, recombinant Ovalbumin (Ova) protein was purchased from Sigma-Aldrich and recombinant influenza hemagglutinin 1 (HA1) was produced as previously described (38 (link)).

Phad G.E., Bernat N.V., Feng Y., Ingale J., Murillo P.A., O’Dell S., Li Y., Mascola J.R., Sundling C., Wyatt R.T, & Karlsson Hedestam G.B. (2015). Diverse antibody genetic and recognition properties revealed following HIV-1 Env immunization. Journal of immunology (Baltimore, Md. : 1950), 194(12), 5903-5914.

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Recombinant HA and NP Protein Expression

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The HA gene was cloned in pFastBac1 and expressed in Spodoptera frugiperdii insect cells, and recombinant HA protein was purified using lentil lectin affinity chromatography (GE healthcare, USA). NP gene cloned in pET15b bacterial expression system was used to transform BL21 codon plus (RIL) cells. Recombinant NP protein was purified using Ni⁺⁺ chelated resin (Invitrogen, USA). Synthetic M2e peptide, SLLTEVETPTRNEWECRCSDSSD, was obtained from INBIOS S.r.l, Italy.

Ingle N.B., Virkar R.G, & Arankalle V.A. (2017). Inter-Clade Protection Offered by Mw-Adjuvanted Recombinant HA, NP Proteins, and M2e Peptide Combination Vaccine in Mice Correlates with Cellular Immune Response. Frontiers in Immunology, 7, 674.

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