Journal: Frontiers in Plant Science

Article Title: In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

doi: 10.3389/fpls.2020.00440

Figure Lengend Snippet: Production of His:hLIF from N. benthamiana leaf tissue extracts by Ni 2+ –NTA affinity column chromatography. (A) Ni 2+ –NTA affinity purification of His:hLIF. Total soluble proteins were extracted from agroinfiltrated N. benthamiana leaf tissue, and recombinant His:hLIF was purified using a Ni 2+ –NTA agarose column with 250 mM imidazole solution as eluent (E). The fractions obtained from purification were analyzed using 12.5% SDS-PAGE followed by Coomassie brilliant blue (CBB) staining. (B, C) Western blot analysis of purified His:hLIF. The His:hLIF elution fractions were separated using 12.5% SDS-PAGE and analyzed by western blotting with anti-LIF (B) or anti-His (C) antibodies. M, molecular weight standard; WT, wild-type N. benthamiana leaf tissue extracts; UB, unbound fraction; W, wash-off solution. The arrows indicate the positions of His:hLIF protein bands at 30 to 42 kDa. (D) Quantification of His:hLIF produced in plants. The concentration of purified His:hLIF was determined using the Bradford protein assay. Samples containing 200 to 350 ng His:hLIF produced in planta or commercial LIF produced in a human cell line were separated using 12.5% SDS-PAGE, and the amounts of each protein were compared using signal intensity from a western blot analysis with anti-hLIF antibody. The concentration of the commercial LIF was provided by the manufacturer. Protein molecular weight standards are marked at the left-hand side.

Article Snippet: His:hLIF was purified using an Ni 2+ –NTA agarose column (Qiagen, CA, USA) according to the manufacturer’s instructions.

Techniques: Affinity Column, Chromatography, Affinity Purification, Recombinant, Purification, SDS Page, Staining, Western Blot, Molecular Weight, Produced, Concentration Assay, Bradford Protein Assay

Journal: Frontiers in Plant Science

Article Title: In Vivo Removal of N-Terminal Fusion Domains From Recombinant Target Proteins Produced in Nicotiana benthamiana

doi: 10.3389/fpls.2020.00440

Figure Lengend Snippet: His:hLIF produced in planta exhibits different degrees of N-glycosylation. Purified His:hLIF was treated with (+) or without (−) endoglycosidase H (Endo-H), and the migration patterns of the treated proteins were analyzed by western blotting with anti-LIF antibody. The arrows indicate N-glycosylated His:hLIF protein bands at 30 to 42 kDa and deglycosylated His:hLIF protein bands at 20 kDa. M, molecular weight standard.

Article Snippet: His:hLIF was purified using an Ni 2+ –NTA agarose column (Qiagen, CA, USA) according to the manufacturer’s instructions.

Techniques: Produced, Purification, Migration, Western Blot, Molecular Weight