03-高效蛋白表达系统.ppt

高效蛋白质表达系统 1. 原核表达系统 (1)Gram negative (E. coli) (2)Gram positive (B. subtilis) 2. 真核表达系统 (1) Yeast (2) Baculovirus (3) Drosophila (4) Mammalian cells (COS, CHO, BHK etc.) 3. 其它 (1) Cell-free system (2) Protoblast Baculovirus Baculovirus are present in invertebrates primarily insect species. They are not infectious for vertebrates plants. Genome is covalently closed circular double stranded of 134 kbp, due to its small it can accommodate large fragments of foreign DNA. They are divided into two groups on the basis of their structure as Nucleopolyhedroviruses (NPV) and Granuloviruses. These NPV are mainly used as expression vectors, i.e. Autographa californica NPV (AcMNPV) isolated from the larva of the alfalfa looper. Baculovirus expression system based upon the ability to propagate AcMNPV in insect cells. Uses many of the protein modification, processing and transport systems present in higher eukaryotic cells. Virus that can be propagated to high titers adapted for growth in suspension cultures obtain large amounts of recombinant protein with relative ease. Baculovirus are noninfectious to vertebrates and their promoters are inactive in mammalian cells. Do and Donts Check cells daily until a confluent monolayer is formed. Passage cells at confluency only, as cells will be easy to dislodge shows better viability. Do not overgrow cells, it results in decreased viability. Do not splits cells too far. Densities lower than 20% confluency inhibit growth. Passage the cells only in log phase, log phase growth can be maintained by splitting cells in 1:5 dilution. The expression of protein complexes Improve the solubility and stability of proteins, especially for big protein products. The tags used. The mutual influences of co-expressed proteins. Example: Bcl-xl/Bim/LC8. OD Volume OD 280 Volume 140KD 68KD ? 21 ? 31 Bcl-xL Bim LC8 ? 14 A B Purification of Bcl-xl/Bim/LC8 complex Drosophila The Drosophila Expression System utilizes a cell line derived from Drosophila melano