Purification of TrxHtt39Q and 3B5H10 Fab Co-complex



TrxHtt39Q in 5 mM Tris pH 8, pI = 5.25, MW = 27095 Da

3B5H10 Fab in 5 mM Tris pH 8, pI = 7.15, MW = 46750 Da

The co-complex pI is 5.9


Day 1:  Making of the co-complex and dialysis

1.   Since the co-complex pI is similar to Htt and different than the Fab, we will make the co-complex in excess of Fab, which will be easily separated in anion exchange chromatography. Make the co-complex in ratio of the following TrxHtt39Q: Fab=1:1.6 (excess 1.6x Fab). Typically 1 run uses 3.5 mg Fab, and 1.3 mg Htt.

2.     Mix the Htt and Fab, and incubate at 4oC for at least 1 hour.

3.   Dialyze the co-complex with 1x 1.5L 20 mM Bis Tris Propane pH 6.3 (titrate with HCl) for overnight at 4oC.


Day 2:  Purification of the co-complex with Anx anion exchange chromatography (2hrs)

1.   Prepare and degas the following buffer:

      Buffer A: 20 mM Bis Tris Propane pH 6.3 (titrate with HCl)

      Buffer B: 20 mM Bis Tris Propane pH 6.3 (titrate with HCl) 1 M NaCl

2.   Centrifuge the sample at 10,000 rpm for 5 minutes, change to fresh tube.

3.   Use HiTrap Anx FF weak anion exchange chromatography (GE Healthcare, 17-5163-01).

      Max flow rate = 5 ml/minutes

      Bed volumes = 5 ml

4.   Prepare the biocad. Connect the 5 ml sample loop. After purging pump A& B with 20% etoh for cleaning, purge pump A and B with the respective buffer A & B. Connect the column to the system. Wash the column with at least 5 column volumes of Buffer A. Wash at 3 ml/minutes flow rate.

5.   Run the program under methods/Kartika/Anx. The following is the outline of the method:

  1. Set the flow rate to 3 ml/minutes
  2. Equilibrate the column in 2 column volumes of buffer A

- Start the fraction collector, collecting 2 ml/fraction

  1. Load the sample in two 3 ml injection
  2. Gradient segment

- Maintain 100% gradient A in 3 column volumes

- Start linear gradient to 100% buffer B in 20 column volumes

- Maintain 100% buffer B in 5 column volumes

- Set to 100% buffer A for 6 column volumes

6.   Do Bradford assay of the fraction, and pool the fraction. Note: The nonbonding peak that elute early is Fab, Fab pI is 7.1, and at pH 6.3 it is positively charge so it does not bind to the anion exchange bead that is also positively charge. While the co-complex peak that elute later, has a pI of 5.9, thus it is negatively charge at pH 6.3 so it bind to positively charge bead. The bound co-complex only elute after the application of salt gradient.

7.   Dialyze the pooled co-complex into 20 mM Bis Tris Propane pH 6.3 (titrate with acetic acid) for overnight at 4oC.




Day 3:  Concentrating the Co-complex

1.   Concentrate the co-complex with Amicon Ultra-4 centrifugal filter device to 600-700 ml volume. Typical concentration is 3.5-4 mg/ml, with the yield about 2.5 mg.