111生物化学理论课件@中科大01绪论2008知识讲稿.ppt

Flow chart: 1, Targets selection: Bioinformatics 2, PCR and cloning: Molecular Biology 3, Protein production: Biochemistry 4, Data collection (X-ray diffraction/NMR) 5,Structure determination: Structural Biology 6, Function interpretation: General Biology Core characteristics: High throughput (hundreds to thousands samples) Large-scale (milligram level of protein sample) Multi-discipline integration (from molecular biology to structural biology) high throughput parallelization (平行化) miniaturization (微量化) automation (自动化) cloning robot: 500-1000 clones /3 days crystallization robot: 96x3 drops/15 min protein sample for crystallization trials in nanoliter volumes (10nl) Hansen CL. et al. PNAS; 2002 Tag Size (aa) Application Sequence or GenBank accession no. His 6 or 8 affinity HHHHHH(HH) T7 11 affinity ASMTGGQQMGR S 15 affinity KETAAAKFERQHMDS Arg 1,5,6 affinity RRRRR(R) CBP 26 affinity KRRWKKNFIAVSAANRFKKISSSGAL FLAG 4 or 8 affinity DYKD or DYKDDDDK Strep 8 affinity WSHPQFEK NusA 491 SET, affinity Accession no. AAN82367 MBP 396 SET, affinity Accession no. AAC43128 GST 220 SET, affinity Accession no. AAB59203 ZZ 116 SET, affinity Accession no. M74186 TrxA 109 SET Accession no. AAC40210 Gb1 56 SET Accession no. 1MPEA DsbA DsbC 208 236 SET SET Accession no. P24991 Accession no. P21892 Zhou CZ Chen YX. Current Genomics; 2004 Different tags will make the difference Different E. coli strains (DE3 series) BL21 (DE3) BL21-pLysS Rosetta Rosetta-pLysS Gold BL21-Codon-plus Tuner Star Co-expression with chaperones (DnaK-DnaJ-GrpE and/or GroEL-GroES) Mainly works for those partially soluble targets Co-purification of chaperones with the target protein Re-aggregation/precipitation after removal of chaperones Eukaryotic expression systems (post-translational modifications) Pichia pastoris Saccharomyces cerevisiae Insect cells Cell lines Virus-mediated Quantity and Cost-effectivity DN