现代生物学ProteinEngineering课件.ppt

The known proteins may not meet human’s needs ;Contents;9.1 PE concept and process;PE process;Comparison;Three specialists are involved: 1) Determination of protein structure 2) Protein molecular design 3) GE;a.a. sequence 一级结构 ;a) X-ray crystallography (晶体X射线衍射分析); Nuclear spin quantum number (核自旋量子数I );NMR ;2D NMR;c) Protein Data Bank (蛋白质结构数据库, PDB) ;X-ray ~80000;Simulation; Design goals;b) de novo design (从头设计) of protein;Only recombinant gene can work! Gene modification gene mutation (site-directed, cassette, PCR) gene fusion gene synthesis et al.;Site-directed mutagenesis (定点突变);Simultaneous generation of 20 mutants with different types of amino acid at one specific site, and saturation analysis (“饱和性”分析) of the important amino acids in the protein;PCR mutation;(b) Gene fusion (融合);Humanized antibodies (“人源化抗体”);First idea: Chimeric antibody (嵌合抗体) Link human C region to mouse V region for Engineered antibody Idea: Retain antigen binding capacity (Keep V region) Solve the heterologous problem (Replace C region) ;Protocol 1 Only replace human CDRs with mouse CDRs Retaining most human V region will greatly eliminate host immune response Closer to human antibody: “humanized antibody” or “reshaped antibody ” (改型抗体);Protocol 2: humanized design of V region;(c) Gene synthesis;Application fields: Life science research, medicine, industry, agriculture, environmental protection, development of fuels;GFP S65T mutant;HIV can spread through cell-cell synapse (“突触”)!;May 28, 2009. Nature 459 (7246) cover Self-inactivating (自身失活) lentiviral (慢病毒) vector (CAG-EGFP) transgenic marmoset (绒猴) The complete gene can be passed and expressed in the offspring ;Advantage： (1) endogenous (内源性), stable, non-toxic (2) Multicolor (多色性) allows simultaneous observation of multiple biomolecules;Transformation of proteins for medical use: stable drug proteins with improved efficacy;Insulin-like growth factor-I (类胰岛素生长因子-I) shows similar 3-D structure with insulin bu