Kim Nan-Sun, Yu Hwa-Young, Chung Nguyen-Duc, Shin Yun-Ji, Kwon Tae-Ho, Yang Moon-Sik
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.
Protein Expr Purif. 2011 Mar;76(1):121-6. doi: 10.1016/j.pep.2010.10.007. Epub 2010 Oct 15.
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.
一种合成牛胰蛋白酶原（sbTrypsinogen）通过重叠PCR策略，基于水稻优化的密码子使用情况进行合成，然后在蔗糖饥饿诱导型水稻α-淀粉酶3D（RAmy3D）启动子的控制下进行表达。利用现有的信号肽实现了胰蛋白酶向培养基中的分泌。通过根癌农杆菌介导，将植物表达载体导入水稻愈伤组织（水稻品种东津）。通过基因组DNA PCR扩增验证了sbTrypsinogen基因整合到转基因水稻愈伤组织的染色体中，并通过Northern印迹分析证实了转基因水稻悬浮细胞中sbTrypsin的表达。Western印迹分析检测到糖饥饿诱导后培养基中存在质量为24至26 kDa的糖基化蛋白。通过明胶酶谱证实了水稻来源的胰蛋白酶的蛋白水解活性，且与商业牛源胰蛋白酶的活性相似。糖饥饿5天后，转基因水稻细胞悬浮培养基中积累的sbTrypsin产量为15 mg/L。
