core domain hpt n-terminal domain h h h h h p h h t h h c-terminal domain h h p h p p h t p p h h t...

Download Core domain HPT N-terminal domain H H H H H P H H T H H C-terminal domain H H P H P P H T P P H H T H P T H T T T P P H H P P H P T H H P H P P P P P T

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  • Slide 1
  • Core domain HPT N-terminal domain H H H H H P H H T H H C-terminal domain H H P H P P H T P P H H T H P T H T T T P P H H P P H P T H H P H P P P P P T P P P H T P P T P T T T T T H H T P H T T H H T H P T P P T T P P T H T T P T T T T T
  • Slide 2
  • Grow cultures to OD=0.6~0.8, add IPTG to 0.2 mM, grow at room temperature for 2~3 hours Collect cells, freeze and thaw for lysis in Buffer IN (20 mM Tris pH8.0, 100 mM NaCl, 5 mM BME, 1 mM PMSF), sonicate briefly to break genomic DNA Centrifuge at 20,000 x g for 30 min at 4 C, separate supernatant and pellet. Resuspend pellet (inclusion body) in Buffer B (20 mM HEPES pH7.5, 1 M NaCl, 10 mM CHAPS, 10% Glycerol, 5 mM BME), stir gently, overnight at 4 C. Centrifuge at 20,000 x g for 30 min at 4 C, collect supernatant for His-tag purification Load on Ni column, wash with buffer HNN (20 mM HEPES pH7.5, 1 M NaCl, 0.1% NP-40, 5 mM BME) + 40 mM Imidazole, elute with HNN+200 mM Imidazole Combine the protein fractions, dialyze in Storage Buffer (20 mM HEPES pH7.5, 0.3 M NaCl, 20% Glycerol, 10 mM DTT, 5 mM CHAPS), store in small aliquots at -80 C
  • Slide 3
  • Core domain HPT N-terminal domain H H H H 0.34 H P H 0.53 H T H 0.54 H C-terminal domain H H P 0.97 H P P 4.12 H T P 0.68 P H H T 2.33 H P T 0.63 H T T 0.78 T P P H H 2.44 P P H 2.63 P T H 1.39 H P H P 1.01 P P P 2.49 P T P 0.75 P P H T 4.00 P P T 2.63 P T T 1.33 T T T H H 0.66 T P H 0.60 T T H 0.71 H T H P 0.59 T P P 0.55 T T P 0.53 P T H T 0.59 T P T 0.45 T T T 0.58 T Conc. in mg/mL
  • Slide 4
  • HXX Proteins (HIV NTD) 26- 55- 43- 34- 70- (kD) HHH HHP HHT HPH HPP HPT HTH HTP HTT M Calc. MW. 34 39 51 34 39 51 35 40 52 30 pmole of each protein were resolved on a 4-20% polyacrylamide gel, coomassie stained.
  • Slide 5
  • PXX Proteins (PFV NTD) 26- 55- 43- 34- 70- (kD) PHH PHP PHT PPH PPP PPT PTH PTP PTT M Calc. MW. 40 45 58 41 46 58 41 46 59 30 pmole of each protein were resolved on a 4-20% polyacrylamide gel, coomassie stained.
  • Slide 6
  • Approaches tested: 13 degree expression To be tested: 4 C expression in lacIq- vector (pET3a). 26- 55- 43- 34- 70- (kD) THH THP THT TPH TPP TPT TTH TTP TTT TXX Proteins (Ty3 NTD) were hard to purify, low purity and concentration (not enough to go through 2 nd round purification)
  • Slide 7
  • IN PCR in vitro integration assay DNA substrate features 3 types of LTR, HIV/PFV/Ty3 Identical left half (one primer detects all.) Overhang vs blunt-end Reaction conditions 250 fmole target DNA plasmids 500 fmole substrate DNA 1 pmole IN 500 fmole TFP when used 23 C or 37 C Mg 2+ or Mn 2+
  • Slide 8
  • Ty3 IN (TTT) strand transfer patterns (previous data) Mg 2+ -----10 mM --- 300 bp 100-200 bp
  • Slide 9
  • DNA substrates mixture, blunt-end, Mn 2+, 37, no TF | TTT | HHH | PPP | PPH | PPT | HHP | 500- 100- DNA substrates can be used as mixture. X
  • Slide 10
  • H Core Proteins Blunt-end Substrates, Mn 2+, 37, no TF HHH HHP HHT PHH PHP PHT THH THP THT H 2 O 500- 100- 500- 100-
  • Slide 11
  • HPH HPP HPT PPH PPP PPT TPH TPP TPT H 2 O P Core Proteins Blunt-end Substrates, Mn 2+, 37, no TF 500- 100- 500- 100-
  • Slide 12
  • HTH HTP HTT PTH PTP PTT TTH TTP TTT H2O 500- 100- 500- 100- T Core Proteins Blunt-end Substrates, Mn 2+, 37, no TF
  • Slide 13
  • Mn 2+ summary H-core proteins showed more robust activity than P- core and T-core. XHH (H-core and H-CTD together) showed higher activity. P-core: only PPP active T-core: only TTT active Inhibitory effect of DNA substrates?
  • Slide 14
  • WT (RT and 37) Overhang Substrates, Mg 2+, with TFP HHH HHH PPP PPP TTT TTT H 2 O RT 37 RT 37 RT 37 500- 100- 500- 100-
  • Slide 15
  • H Core Overhang Substrates, Mg 2+, RT, with TFP HHH HHP HHT PHH PHP PHT THH THP THT H2O 500- 100- 500- 100-
  • Slide 16
  • P Core Overhang Substrates, Mg 2+, RT, with TFP HPH HPP HPT PPH PPP PPT TPH TPP TPT H 2 O 500- 100- 500- 100-
  • Slide 17
  • T Core Overhang Substrates, Mg 2+, RT, with TF HTH HTP HTT PTH PTP PTT TTH TTP TTT H 2 O 500- 100- 500- 100-
  • Slide 18
  • Effect of TFP on selected proteins Overhang Substrates, Mg 2+, RT TTT HTT PTH PTP HHP PHH TTT HTT PTH PTP HHP PHH H 2 O With TF Without TF 500- 100- Assay not working.
  • Slide 19
  • Fresh thaw of IN aliquots Overhang Substrates, Mg 2+, RT TTT HTT PTH PTP HHP PHH TTT HTT PTH PTP HHP PHH H 2 O With TF Without TF
  • Slide 20
  • New 2x Mg reaction Buffer Overhang Substrates, Mg 2+, RT TTT HTT PTH PTP HHP PHH TTT HTT PTH PTP HHP PHH H 2 O With TFWithout TF
  • Slide 21
  • Compare individual and mixed DNA substrates Purify Ty3 N-terminal proteins, 4 C expression. Assay conditions. Future experiments

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