MBP-TPH AC w/ on-column cleavage - MBP-TPH GF

Purification of recombinant MBP-chTPH1 (HC3440)
The purpose of this project is to purify the catalytic domain of human tryptophan hydroxylase isoform 1 (chTPH1). Although it is an iron-containing enzyme it is colourless, so you only have the 280 nm absorbance to follow the protein. The product of this purification is the protein of interest without the purification tag (MBP). This is achieved by on-column cleavage using a recombinant MBP-tagged human rhinovirus (HRV) type 14 3C protease,  known as 3CP. The MBP-3CP is expressed in E. coli and purified. It is provide ready by DTU Chemistry staff. 3CP cleaves a specific amino acid sequence (LeuGluValLeuPheGln ↓ GlyPro) and can be used to cleave fusion tag sequences from recombinant proteins that contain an HRV 3C protease cleavage site. The amino acid sequences for MBP-chTPH1 and chTPH1 can be found at the bottom of this document.

A short introduction to tryptophan hydroxylase can be found on these links:

wikipedia - Tryptophan hydroxylase  
wikipedia - TPH1

                                  

Buffers prepared for you:

Binding+gelfiltration buffer: 2.5 l 20mM Tris/H2SO4, 50mM (NH4)2SO4, pH 8.0

Regeneration solutions:
                        1.5 l Degassed Ultra-Pure water,

0.2 l 0.5 M NaOH.

1.5 l 20 % v/v ethanol.

Buffers you need to prepare:

Elution buffer: 250 ml 20 mM Tris/H2SO4, 50mM (NH4)2SO4, 1mM maltose, pH 8.0

     Equilibration of gel filtration column (the first class only):

  • Flush buffers on ÄKTA Start using Ultra-Pure water as buffer B and gel filtration buffer as buffer A.

  • Attach a 26/600 Superdex 75 pg Hiload column (flow rate: 1.0 ml/min, pressure limit 0.30 MPa).

  • Fill the flask containing gel filtration buffer to maximum.

  • Run method Gel start stop.

    Purification day one:

  1. Resuspend cells from one 650 mL culture with binding buffer to a volume of 40 ml.

  2. Lyse the cells by sonication (in Satorius Labsonic P, 80% amplitude, 1 cycle) 3x30 sec on wet ice.

  3. Transfer 2x20-µl suspension for SDS-Page to micro centrifuge tubes. Name them R for resuspension.

  4. Centrifuge the protein suspension at 4°C and 12000 rpm (≈18500 g) for 20 minutes and decant supernatant to a new centrifuge tube.
    Resuspend the pellet with 40 ml binding buffer and transfer 2x20-µl sample to micro centrifuge tubes. Name them P for pellet.

  5. Centrifuge the supernatant at 4°C and 12000 rpm (≈18500 g) for 20 minutes. Decant supernatant to a new centrifuge tube. Keep protein on ice.
    Transfer 2x20-µl sample to micro centrifuge tubes. Name them S for supernatant.

  6. Turn on ÄKTA Start, flush buffers, and attach a 16/10 Dextrin Sepharose column (flow rate: 1.0 ml/min).

  7. Start equilibration of the column (flow rate: 5.0 ml/min, pressure limit 0.30 MPa). 5 CV binding buffer. Check that UV is stable.

  8. Filter the supernatant through a 0.45µm syringe filter.

  9. Prepare the protease solution by diluting MBP-3CP stock in binding buffer (use a 50 ml centrifuge tube), to a concentration of 2.2 µM and a total volume of 35 ml (app. 1 tube). Filter through 0.45µm filter.

  10. Load the supernatant onto the column (flow rate: 5.0 ml/min, pressure limit 0.30 MPa). Wash with 5 CV of binding buffer. Collect 1 5 ml fraction from the flow through and transfer 2x20-µl sample for SDS-page to micro centrifuge tubes. Name them FT.

  11. Load 30 ml of protein solution to the column. Pause purification for 1 hour (cleavage time).

  12. Start wash with 5 CV binding buffer. Start collecting in 5 ml fractions after 8 ml of wash. Stop fractionating when protein stops eluting.

  13. Collect the first 1 or 2 fractions and pool them together. Keep on ice. This is your target protein. Transfer 2x20-µl sample for SDS-page to micro centrifuge tubes. Name them TP.

  14. Elute uncleaved protein with 5 CV elution buffer. Collect protein in 5 ml fractions. Transfer 2x20-µl sample for SDS-page to micro centrifuge tubes. Name them E.

  15. Measure the protein concentration on the nanodrop at 280 nm (note volume).

  16. Start regeneration of the Dextrin Sepharose using a peristaltic pump (flow rate: 2.5 ml/min).

  17. Flush buffers on ÄKTA Start and attach a 26/600 Superdex 75 pg Hiload column.

  18. Filter the protein through a 0.45µm syringe filter.

  19. Load a maximum of 13 ml protein solution onto the column (flow rate: 2.6 ml/min, pressure limit 0.30 MPa). Elute and collect the target protein isocratically using 1 CV gel filtration buffer.

  20. Collect eluting protein in 5 ml fractions. Transfer 2x20-µl sample for SDS-page to micro centrifuge tubes from each peak. Name them G1, G2…

  21. Measure the protein concentration on the nanodrop at 280 nm (note volume).

  22. Concentrate chTPH1 using a Sartorius Vivaspin Turbo centrifuge concentrator fitted with a NMWL 5 or 10 kDa membrane.

  23. Measure the protein concentration on the nanodrop at 280 nm (note volume).

  24. Transfer protein to a 15 ml centrifuge tube and freeze in liquid nitrogen (see how here link: Storage of protein samples in liquid nitrogen) then store in -80°C freezer.

  • Store SDS-Page samples at -20°C.

     

Regeneration of Dextrin Sepharose columns

  1. Regenerate column using 3 CV Ultra-Pure water, 3 CV 0.5 M NaOH, 4 CV Ultra-Pure water, check that pH is equal to or below 7, 3 CV 20 % v/v ethanol. Store column in refrigerator.

    Reequilibration of gel filtration column (the second class only):

  2. Flush buffers on ÄKTA Start using Ultra-Pure water as buffer B and 20 % v/v ethanol as buffer A.

  3. Attach a 26/600 Superdex 75 pg Hiload column (flow rate: 1.0 ml/min, pressure limit 0.30 MPa).

  4. Fill the flask containing 20 % v/v ethanol to maximum.

  5. Run method Gel start stop.

    MBP-chTPH1

    MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRY
    NGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKN
    KHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGA
    VALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGLEVLFQGPDGMETVPWF
    PKKISDLDHCANRVLMYGSELDADHPGFKDNVYRKRRKYFADLAMNYKHGDPIPKVEFTEEEIKTWGTVFQELNKLYPTHACREYLKNLPLLSKYCGYRE
    DNIPQLEDVSNFLKERTGFSIRPVAGYLSPRDFLSGLAFRVFHCTQYVRHSSDPFYTPEPDTCHELLGHVPLLAEPSFAQFSQEIGLASLGASEEAVQKLA
    TCYFFTVEFGLCKQDGQLRVFGAGLLSSISELKHALSGHAKVKPFDPKITCKQECLITTFQDVYFVSESFEDAKEKMREFTKTIKRPFGVKYNPYTRSIQILKD

    chTPH1
    GPDGMETVPWFPKKISDLDHCANRVLMYGSELDADHPGFKDNVYRKRRKYFADLAMNYKHGDPIPKVEFTEEEIKTWGTVFQELNKLYPTHACREYLKNL
    PLLSKYCGYREDNIPQLEDVSNFLKERTGFSIRPVAGYLSPRDFLSGLAFRVFHCTQYVRHSSDPFYTPEPDTCHELLGHVPLLAEPSFAQFSQEIGLASLG
    ASEEAVQKLATCYFFTVEFGLCKQDGQLRVFGAGLLSSISELKHALSGHAKVKPFDPKITCKQECLITTFQDVYFVSESFEDAKEKMREFTKTIKRPFGVKYN
    PYTRSIQILKD

 
Storage of protein samples in liquid nitrogen

Please make sure you have made an appropriate risk assessment and have all the necessary safety protection gear at hand, inclusive dioxygen monitor and alarm.

An introduction to different way of storage protein samples can be found here.

This video shows what you have to do.

This video shows what NOT to do. Please be aware that liquid nitrogen is a major hazard and any attempt to do the experiments shown in the video will result in immediate dismissal from the course.

What happens to salts and buffers in the solution if you freeze slowly?

Opdateret den 25. maj 2018
Opdateret den 25. maj 2018