Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures.
A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.
Division of Biological Sciences and Institute for Molecular Biology and Genetics, Chonbuk National University, 664-14 Dukjindong 1Ga Dukjingu, Jeonju, Jeonbuk 561-756, Republic of Korea.
Nan-Sun
NS
Kim
Hwa-Young
HY
Yu
Nguyen-Duc
ND
Chung
Yun-Ji
YJ
Shin
Tae-Ho
TH
Kwon
Moon-Sik
MS
Yang
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D012275:Oryza; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014357:Trypsin / Q000096:biosynthesis*; D014362:Trypsinogen / Q000096:biosynthesis
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D014362:Trypsinogen; D014357:Trypsin