Hi,
I am wondering if anyone know what causes it. I
am trying to express a mouse hormone by either:
1) MBP fusion using pMal-c2x. 2) GST fusion
using pGEX-2T. The mouse cDNA also carries a C-
terminal Histidine tag.
For the application of 1 and 2, I got some good
yield using affinity column against MBP or GST.
But when I look at the SDS-PAGE, 70% of the
purified protein are the size of MBP or GST (2
bands, one is the size of MBP or GST anf the
other is MBP or GST fusion protein). If I
purified the recombinant protein with Histidine
tag, I got only one single band (MBP or GST
fusion) on SDS-PAGE but the yield is kind of low
for our expectation (200~300 microgram per liter).
Does anyone know how to increase the full length
expression of fusion protein? I have tried to
change the expression temperature( from 14 to 37
degree), inducing time (3~8 h for 37 C, overnight
for 14 C) and the results are quite similar. I
need to get the hormone in gram scale so I can do
in vivo injection study.
Thanks,
Remi
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