Lecture14 - MCB110L Lecture 14 03/04/11 Andreas Martin...

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MCB110L Lecture 14 03/04/11 Andreas Martin
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Expression of Cin8p in E.coli : plasmid used in class is pET24b pET = RNA-polymerase expression system, highly selective for bacteriophage T7 RNA polymerase (Studier and Moffatt 1986) T7 polymerase inserted in host chromosome, under control of an IPTG-inducible promoter ( P lac ) often inserted as λ -DE3 = bacteriophage λ carrying T7 Pol gene lac lacO T7 RNA pol. lacI Addition of IPTG induces expression of the T7 RNA polymerase, which then can bind the T7 promoter ( T7 ) in pET and transcribe the gene of interest For even tighter expression (very low levels before IPTG induction) use an additional plasmid pLysS that codes for T7 lysozyme. -> T7 lysozyme represses low-level expression by destabilizing the open complex during transcription initiation pET ~ 5.2 kB kan R T7 your gene of interest BL21( DE3 )
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Protein: yeast Cin8, condon usage table: E.coli K12 Y axis: relative adaptiveness gray <20% red <10% Certain codons for R, L, and I are rare in E.coli Heterologous protein expression : consider codon usage / potential codon biases graphical codon usage analyzer: http://gcua.schoedl.de/
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pGroRIL (Cm R ) GroEL GroES ArgU P ara IleY LeuW coding for rare tRNAs (codons AGA, AGG; AUA; CUA) constitutively expressed for class: additional plasmid in the expression strain Codon biases can strongly affect protein expression levels -> expression of the rare tRNAs in E.coli (Stratagene) expression of 3 eukaryotic genes (A,B,C) that are affected by codon bias
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Strong protein overexpression can lead to misfolding and aggregation especially a problem for heterologous expression of eukaryotic proteins in prokaryotes, e.g. E.coli no formation of S-S bonds in the cytosol no post-translational modifications in prokaryotes k f k a k a competition of folding and aggregation: k f = 1 st order rate constant, f(conc.) k a 2 nd order, conc.-dependent but aggregation can also be beneficial: formation of inclusion bodies (IB) extreme overproduction (> 1 g/L culture) phase separation, no interference with cell metabolism high density -> easy purification protected from proteolysis prerequisite : after IB solubilization with denaturant (GdmCl, urea), the protein has to be able to refold in vitro with IB
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Molecular chaperones: proteins that assist folding (or unfolding) and the assembly (or disassembly) of other macromolecular structures, but are not part of these structures prevent aggregation often heat shock proteins (Hsp) , expressed in response to elevated T or other stresses that cause to protein unfolding some chaperones specialized for folding newly-made proteins at ribosomes avoiding protein aggregation during expression: lower expression rate (plasmid copy number, weaker promoters, etc.) express at lower temperatures express eukaryotic proteins in eukaryotic cells (insect cell) co-expression of molecular chaperones action of chaperones
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http://movingscience.de/en/projects/biology/chaperone_assisted_protein_folding/video.html
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This note was uploaded on 09/12/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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Lecture14 - MCB110L Lecture 14 03/04/11 Andreas Martin...

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