Bösze Zsuzsanna, Hiripi László
Agricultural Biotechnology Center, Gödöll, Hungary.
Methods Mol Biol. 2012;824:629-41. doi: 10.1007/978-1-61779-433-9_34.
Producing complex recombinant proteins in the milk of transgenic animals offers several advantages: large amounts of proteins can be obtained, and in most cases, these proteins are properly folded, assembled, cleaved, and glycosylated. The level of expression of foreign genes in the mammalian gland cannot be predicted in all cases, and appropriate vectors must be used. The main elements of these vectors are as follows: a well-characterized specific promoter, the coding region of the gene of interest, preferably with a homologous or heterologous intron, to improve transcription efficiency, and an insulator or boundary element to counteract the chromosomal position effects at the integration site. Once high expression levels are achieved, and the recombinant protein is purified, an essential step in the analysis of the final product is determining its degree of glycosylation. This is an important readout because it can affect among other parameters the stability and immunogenicity of the recombinant protein.
可以获得大量蛋白质，并且在大多数情况下，这些蛋白质能正确折叠、组装、切割和糖基化。并非在所有情况下都能预测外源基因在哺乳动物乳腺中的表达水平，因此必须使用合适的载体。这些载体的主要元件如下：一个特征明确的特异性启动子、目的基因的编码区，最好带有同源或异源内含子以提高转录效率，以及一个绝缘子或边界元件以抵消整合位点处的染色体位置效应。一旦实现高表达水平并纯化出重组蛋白，分析最终产物的一个关键步骤就是确定其糖基化程度。这是一个重要的指标，因为它会影响重组蛋白的稳定性和免疫原性等参数。
