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1、Chapter 8Gene Cloning and Expression8.4 Gene cloning and gene expression8.4 Gene cloning and gene expression Whole-genome sequencing Easy to culture Reproduction rapidly Easy to handle Genetic stability Advantages of EAdvantages of E.coli expression system.coli expression system Target gene Choice o
2、f vector Ligation of target gene and vector Host bacteria strains and induction conditions8.4.1 The basic elements of cloned gene expression in E.coliThe target gene must be cDNAif it comes from eukaryotic cells.The upstream part of the initiation codon of cDNA(5 non-coding region)is useless and mus
3、t be removed.For somesecretory proteins,signal peptides should also be removed.(1)Target geneThe expression vector used must be an E.coliexpression vector,containing a promoter and SDsequence.(2)Choice of vector Initiation codon Expression of fusion protein(3)Ligation of target gene and vectorAn Nde
4、 l or Nco I site was constructed downstream ofthe 3 end of the SD sequence in some vectors.After thetarget gene is sheared,the ATG in the Nde I or Nco Isitecanbeusedastheinitiationcodon.Thisconstruction method is suitable for expressing non-fusion proteins in E.coli.Initiation codonThe SD sequence o
5、f some vectors is followed by astructural gene of E.coli protein.The 3 end of thisgene contain a multiple cloning site,which isconvenient for the insertion of the target gene.Aftertranscription and translation,the fusion proteincan be generated.Expression of fusion protein Host bacteria:Not all bact
6、eria strains can obtainthe same expression efficiency for a specific protein.Induction conditions:conditions required for theinduced expression of different foreign genes arenotalwaysthesame,actuallytheoptimalconditions should be explored.(4)Host(4)Host bacteria and induction bacteria and induction
7、conditions conditions Improve the level of translation;Separate the growth of bacteria from the expression offoreign genes;Improve the stability of expressed protein.8.4.2 Increase the expression level of foreign gene Adjust the distance between the SD sequenceand the initial codeATG Change some bas
8、es by point mutation method Increase the stability of mRNA(1)Improve the translation levelDividing the growth of host and the expression offoreigngenesintotwostagesisthemostcommonly used method.（2）Separate the growth of bacteria from the expression of foreign genesTemperature-inducedDrug-induced Exp
9、ress fusion protein use of a certain mutant strain Express the secretory protein（3）Improve the stability of expressed protein Fusion protein is relatively stable and not easy to be hydrolyzed;If the structural gene of E.coli is a signal peptide,it can producesecretory products;The prokaryotic protei
10、n part can be cut off by protease to releaseeukaryotic protein.Advantages:Advantages:Express fusion proteinThe synthesis of protease mainly depends onhypoxanthinenucleoside,hencetheuseofhypoxanthine nucleoside deficient strains as therecipient bacteria can inhibit the synthesis ofprotease in E.coli,protect the expressed proteinfrom degradation.use of a certain mutant strainThank you!