Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C

• Published in: Enzyme and microbial technology 2018-01, Vol.108, p.66-73
• Main Authors: Islam, Md Reyazul, Kim, Nan-Sun, Jung, Jae-Wan, Kim, Hyo-Boon, Han, So-Chon, Yang, Moon-Sik
• Format: Article
• Language: English
• Subjects: Agrobacterium tumefaciens - genetics; Amino Acid Sequence; Animals; Cattle; Cell Line; Enzyme Activation; Genetic Vectors; Hemoglobins - metabolism; Human pepsin C; Human pepsinogen C (hPGC); Humans; Hydrogen-Ion Concentration; Kinetics; Oryza - genetics; Oryza - metabolism; Pepsin A - chemistry; Pepsin A - genetics; Pepsin A - metabolism; Pepsinogen C - chemistry; Pepsinogen C - genetics; Pepsinogen C - metabolism; Plants, Genetically Modified; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rice cell suspension culture; Rice α-amylase 3D promoter; Substrate Specificity; Temperature
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2017.09.006

•We constructed transgenic rice cell suspension culture producing synthetic human pepsinogen C (shPGC).•The expression of shPGC was determined by Northern and Western blot analyses.•The accumulated hPGC and its mature form, human pepsin C in the culture medium was 18mg/L on day 5.•The human pepsin C was purified using a Ni-NTA column under pH 2.0.•Rice-derived pepsin activity was showed structural and biochemical properties similar to those of commercial pepsin. A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3′ end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.