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...

Core domain H P T

N-terminal domain

HH H H   H P H   H T H   H

C-terminal domain

H H P   H P P   H T P   PH H T   H P T   H T T   T

PP H H   P P H   P T H   HP H P   P P P   P T P   PP H T   P P T   P T T   T

TT H H   T P H   T T H   HT H P   T P P   T T P   PT H T   T P T   T T T   T

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

Core domain H P T

N-terminal domain

HH 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 PH H T 2.33 H P T 0.63 H T T 0.78 T

PP H H 2.44 P P H 2.63 P T H 1.39 HP H P 1.01 P P P 2.49 P T P 0.75 PP H T 4.00 P P T 2.63 P T T 1.33 T

TT H H 0.66 T P H 0.60 T T H 0.71 HT H P 0.59 T P P 0.55 T T P 0.53 PT H T 0.59 T P T 0.45 T T T 0.58 T

Conc. in mg/mL

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.

PXX Proteins (PFV NTD)

26-

55-43-34-

70-

(kD) PHH

PHP

PHT

PPH

PPP

PPT

PTH PT

PPTT

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.

Approaches tested:•  13 degree expressionTo 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 2nd round purification)

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

• Mg2+ or Mn2+

Ty3 IN (TTT) strand transfer patterns (previous data)

Mg2+ -----10 mM ---

300 bp 100-200 bp

DNA substrates mixture, blunt-end, Mn2+, 37°, no TF

| TTT | HHH | PPP | PPH | PPT | HHP |

500-

100-

DNA substrates can be used as mixture.

X

H Core Proteins Blunt-end Substrates, Mn2+, 37°, no TF

HHH HHP HHT PHH PHP PHT THH THP THT H2O

500-

100-

500-

100-

HPH HPP HPT PPH PPP PPT TPH TPP TPT H2O

P Core Proteins Blunt-end Substrates, Mn2+, 37°, no TF

500-

100-

500-

100-

HTH HTP HTT PTH PTP PTT TTH TTP TTT H2O

500-

100-

500-

100-

T Core Proteins Blunt-end Substrates, Mn2+, 37°, no TF

Mn2+ 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?

WT (RT and 37°)Overhang Substrates, Mg2+, with TFP

HHH HHH PPP PPP TTT TTT H2ORT 37 RT 37 RT 37

500-

100-

500-

100-

H CoreOverhang Substrates, Mg2+, RT, with TFP

HHH HHP HHT PHH PHP PHT THH THP THT H2O

500-

100-

500-

100-

P CoreOverhang Substrates, Mg2+, RT, with TFP

HPH HPP HPT PPH PPP PPT TPH TPP TPT H2O

500-

100-

500-

100-

T CoreOverhang Substrates, Mg2+, RT, with TF

HTH HTP HTT PTH PTP PTT TTH TTP TTT H2O

500-

100-

500-

100-

Effect of TFP on selected proteinsOverhang Substrates, Mg2+, RT

TTT HTT PTH PTP HHP PHH TTT HTT PTH PTP HHP PHH H2OWith TF Without TF

500-

100-

Assay not working.

Fresh thaw of IN aliquotsOverhang Substrates, Mg2+, RT

TTT HTT PTH PTP HHP PHH TTT HTT PTH PTP HHP PHH H2O

With TF Without TF

New 2x Mg reaction BufferOverhang Substrates, Mg2+, RT

TTT HTT PTH PTP HHP PHH TTT HTT PTH PTP HHP PHH H2O

With TF Without TF

•Compare individual and mixed DNA substrates

•Purify Ty3 N-terminal proteins, 4 C expression.

•Assay conditions. 

Future experiments