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Cold Shock Vectors

Cold Shock Vectors. Optimal Growth Acclimation Steady Low Temp Growth Stationary 37ºC Phase Adapted Cells Phase. CSPs. Growth Curve. Non-CSPs. CSPs. Non-CSPs. Induction of cold-shock proteins in Esherichia coli. 37 °C. 15. , 30 min.

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Cold Shock Vectors

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  1. Cold Shock Vectors

  2. Optimal GrowthAcclimation Steady Low Temp Growth Stationary 37ºCPhase Adapted Cells Phase CSPs Growth Curve Non-CSPs CSPs Non-CSPs

  3. Induction of cold-shock proteins in Esherichia coli 37°C 15 , 30 min 15 °C , 1 hr °C

  4. ▪ Protein Unfolding ▪ Protein Degradation Cold shock Heat shock ▪ RNA Stabilization RNA Chaperones Molecular Chaperones ▪ Ribosome Dysfunction RbfA, CsdA Proteases ▪ mRNA Degradation PNPase

  5. W11 The primary and tertiary structure of CspA -4  -5 -2 -1 MSGKMTGIVKWFNADKGFGFITPDDGSKDVFVHFSAIQNDGYKSLDEGQKVSFTIESGAKGPAAGNVTSL RNP2 RNP1 F12 Y42 F20 F53 F34 F18 H33 F31

  6. The molecule number of CspA homologues in cold-shocked cells Cold shock time (hr) Purified CspA protein (ng) 0 1 3 6 1 2 4 8 16 32 64 128 37ºC 15ºC C I C I E B A G E B A G CspA : 1 × 106 molecules/cell ( CspA + B + G + E : 2 × 106 molecules/cell )

  7. Effect of nonsense mutations AUG A cspA promotor 5’-UTR SD coding sequence 3’-UTR * pJJG02 * * pA01S * * pA10S * * pA30S pUC19 pJJG02 pA01S pA10S pA30S B C 5 0 1 3 6 hr 0 1 3 6 0 1 3 6 CS: 0 1 3 6 01 3 6 pUC19 4.5 pJJG02 4 pA01S pA10S 3.5 pA30S 3 OD600 2.5 2 1.5 1 0.5 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Time after cold shock (hr)

  8. Ribosomal profiles of cells carrying the mutant plasmids C A pA10S pJJG02 D B pA30S pA01S

  9. LACE LowTemperature-dependentAntibiotic Effect of Truncated cspAExpression Ribosome Trapping

  10. Cold-Shock Vector Single-Protein Synthesis at low temperature without producing any other cellular proteins NMR without protein purification

  11. cspA 3’-UTR BamHI KpnI HindIII NdeI SalI Multiple cloning site Factor Xa cleavage site [His]6 cspA ORF Ap pCold (prototype) 3100bp cspA 5’-UTR T7/cspA promoter ori

  12. Induction at 15 oC with pCold I Induction at 37 oC with a T7 vector (3 hr) _______ ______ ______ _______ _______ 0 12 24 0 12 24 0 12 24 0 12 24 0 12 24 hr - + - + - + - + - + ____ ____ ____ _____ _____ WR:49 35 26 27 53 WR49 WR35 WR26 WR27 WR53 Production of C. elegans Proteins

  13. -216 kDa -132 -45.7 -32.5 -18.4 _________ __________ _________ 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 days WR49 WR35 WR26

  14. Expression of γINF in pcold II vector -interferon 0 1 3 6 hr at 15° C 1 mM IPTG

  15. A B cspA 3’-UTR cspA 3’-UTR BamHI BamHI NdeI NdeI Factor Xa cleavage site DB [His]6 cspA 5’-UTR cspA ORF Ap pCold I (3100bp) Ap pCold II (2900bp) cspA promoter cspA 5’-UTR cspA promoter ori ori C cspA 3’-UTR BamHI NdeI cspA 5’-UTR Ap pCold III (2900bp) cspA promoter ori

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