Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant

Author:

Kim Kibum1,Kang Yang Joo1ORCID,Park Se Ra1ORCID,Kim Do-Sun2,Lee Seung-Won1,Ko Kinarm3,Ponndorf Daniel4,Ko Kisung1

Affiliation:

1. Department of Medicine, Therapeutic Protein Engineering Lab, College of Medicine, Chung-Ang University, Seoul, South Korea

2. Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun, South Korea

3. Department of Stem Cell Biology, Konkuk University School of Medicine, Konkuk University, Seoul, South Korea

4. Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Colney, Norwich, UK

Abstract

Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-FcK was infiltrated into the leaves of Nicotiana benthamiana plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1–10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated N. benthamiana leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.

Funder

Korean Rural Development Administration

Korean Government

Publisher

PeerJ

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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