Eskelin Katri, Ritala Anneli, Suntio Taina, Blumer Susan, Holkeri Heidi, Wahlström Eva H, Baez Julio, Mäkinen Kristiina, Maria Nuutila Anna
Department of Applied Chemistry and Microbiology and Department of Applied Biology, University of Helsinki, Helsinki, Finland.
Plant Biotechnol J. 2009 Sep;7(7):657-72. doi: 10.1111/j.1467-7652.2009.00432.x. Epub 2009 Jul 27.
Recombinant DNA technology can be used to design and express collagen and gelatin-related proteins with predetermined composition and structure. Barley seed was chosen as a production host for a recombinant full-length collagen type I alpha1 (rCIa1) and a related 45-kDa rCIa1 fragment. The transgenic barley seeds were shown to accumulate both the rCIa1 and the 45-kDa rCIa1 fragment. Even when the amount of the rCIa1 was just above the detection threshold, this work using rCIa1 as a model demonstrated for the first time that barley seed can be used as a production system for collagen-related structural proteins. The 45-kDa rCI1a fragment expression, targeted to the endoplasmic reticulum, was controlled by three different promoters (a constitutive maize ubiquitin, seed endosperm-specific rice glutelin and germination-specific barley alpha-amylase fusion) to compare their effects on rCIa1 accumulation. Highest accumulation of the 45-kDa rCIa1 was obtained with the glutelin promoter (140 mg/kg seed), whereas the lowest accumulation was obtained with the alpha-amylase promoter. To induce homozygosity for stable 45-kDa rCIa1 production in the transgenic lines, doubled haploid (DH) progeny was generated through microspore culture. The 45-kDa rCIa1 expression levels achieved from the best DH lines were 13 mg/kg dry seeds under the ubiquitin promoter and 45 mg/kg dry seeds under the glutelin promoter. Mass spectroscopy and amino acid composition analysis of the purified 45-kDa rCIa1 fragment revealed that a small percent of prolines were hydroxylated with no additional detectable post-translational modifications.
重组DNA技术可用于设计和表达具有预定组成和结构的胶原蛋白及明胶相关蛋白。大麦种子被选为重组全长I型胶原蛋白α1（rCIa1）和相关的45 kDa rCIa1片段的生产宿主。转基因大麦种子被证明能积累rCIa1和45 kDa rCIa1片段。即使rCIa1的量刚高于检测阈值，以rCIa1为模型的这项工作首次证明大麦种子可作为胶原蛋白相关结构蛋白的生产系统。靶向内质网的45 kDa rCI1a片段的表达由三种不同的启动子（组成型玉米泛素启动子、种子胚乳特异性水稻谷蛋白启动子和萌发特异性大麦α淀粉酶融合启动子）控制，以比较它们对rCIa1积累的影响。谷蛋白启动子使45 kDa rCIa1的积累量最高（140 mg/kg种子），而α淀粉酶启动子使积累量最低。为了在转基因系中诱导纯合性以稳定生产45 kDa rCIa1，通过小孢子培养产生了双单倍体（DH）后代。在泛素启动子下，最佳DH系的45 kDa rCIa1表达水平为13 mg/kg干种子，在谷蛋白启动子下为45 mg/kg干种子。对纯化的45 kDa rCIa1片段进行质谱分析和氨基酸组成分析表明，只有一小部分脯氨酸被羟基化，未检测到其他翻译后修饰。
