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Fig. 1 | Plant Methods

Fig. 1

From: Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

Fig. 1

Variable lymphocyte receptors as tools to target plant-expressed proteins. A schematic diagram depicting the steps involved in developing an LRR-containing VLR that binds to plant-expressed proteins. (1) Express and purify the protein from E. coli, P. pastoris, or other sources. (2) Immunize lampreys with the purified protein of interest conjugated to an adjuvant for the production of VLRB antibodies. (3) Clone VLRBs from lamprey’s lymphocytes into a yeast surface display (YSD) library. (4) Enrich the YSD library for high-affinity binding VLRBs using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS), and identify individual high-affinity binding VLRBs using flow cytometry. (5) Clone VLRBs into plant expression vectors for in planta expression. The LRR-containing VLR may be modified to carry additional modules (e.g., enzymes or receptors). Step 1 shows Denville Blue™ staining of SDS-PAGE gel of E. coli expressed His6-HopM11–300. (A) Ni–NTA agarose purified protein. (B) Anion-exchange chromatographic flow-through. (C) Fraction eluted with 433 mM NaCl from the anion exchange chromatographic column, which after dialysis into phosphate buffer was used to inoculate lampreys for VLRB production. Step 4 shows the YSD library before enrichment for VLRBs that bind HopM11–300 with high affinity (non-sorted), and after MACS and FACS selection

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