mol9 - Lecture 9 – Expression of Recombinant...

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Unformatted text preview: Lecture 9 – Expression of Recombinant Proteins these slides from Lecture 7 (finshing Ch. 5) Review/help schedule Next slide and WebCT Midterm Exam in 2 locaJons Center 276 Bird’s Eye View Heading to BIMM 100 Mid ­Term Mon Tues Wed Thur Fri Sat Sun 8 am 22 23 Noon 1 pm 24 25 11 am 2 pm Lecture 8 PracJce MidTerm Posted @ WebCT 26 9:30 OH 2 ­4 Q&A Pac Hall 3501 & 3502 2 SF 1 pm 1 RC 27 Lecture 9 28 8 ­10 AY 11 am OH Noon 2 CE 4 SG 7 ­9 p.m. Review Pepper Canyon 109 11 am Noon 12:20 AG 12:30 TG 29 8 am 9:30 Exam Peterson 108 & Center 276** 3 ­5 Q & A Pac Hall 3501 & 3502 6:30 RY 7 pm SecJons Office Hours (TA) Office Hours (Prof Pillus) Special Events See Master Schedule for SecJon & OH LocaJons (or confirm special mtgs with TA indicated by iniJals) ** Students taking exam in Center 276: Abihider, Katherine – Ho, Henry cloning! finding a clone/gene! defining (describing/sequencing) a clone! PCR ! learning from hybridization: blots, in situs, arrays .......! knockouts – making mutants to define function! expression cloning! vectors (plasmids)! restriction endonucleases, DNA ligases! poly-linkers! libraries (oligos)! making high levels of proteins from cloned genes (cDNAs)! general strategies and considerations! some success stories! potential problems to consider! What to make?! What host cells?! How to purify?! Efficiency and Economy ! expressing a cloned cDNA! general strategies and considerations! a ʻclassicʼ inducible system from E. coli! MCB 5-31! choice of promoters /inducers allows ! experimental control of expression! expressing a cloned cDNA! MCB 5-31! choose desired product! subclone - a generic term! (move into optimal vector)! transform into host cell! optimize growth ! and induction conditions ! Tricks: engineer ʻtagsʼ! to simplify purification! purify / process! product! Some success stories -- important products! Product ! ! ! ! !Purpose! restriction endonucleases ! clotting factor VIII! clotting factor IX ! erythropoietin insulin ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !more cloning!! !hemophilia A! !hemophilia B! !anemia $$ ! !type I diabetes! !gh deficiencies $$! !prevention! !hairy cell leukemia;! !chronic hepatitis! !multiple sclerosis! !cystic fibrosis! growth hormone ! hepatitis B vaccine α-interferon ! ! ! ! β-interferon ! DNase ! ! ! ! ! ! some potential (or realized) problems! expression (toxic to host cell)! purification! immunogenic! inappropriate use$$ ! not fully functional! modification, processing! folding …..! Expression in animal cells! Clone gene of interest in shuttle vector! Introduce into host cells! Choices:! Transient vs. Stable! Cell type! Induction system! (promoters and/or inducers)! Animal cells do not! stably replicate and ! segregate plasmids! MCB 5-32! Tags - (epitopes -if corresponding antibody)! sequences encoding peptides/! proteins not found in host cell! (see 5-33) Check your understanding!! Compare: Southern, Northern and analysis,! Microarrays, ʻin situsʼ! What material is being analyzed?! What is the probe?! What information will you learn?! What controls do you need?! Knockouts: How? Compare strategies EssenJal vs. non ­essenJal genes Expression cloning! What do you need?! Potential problems?! ...
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