Hi,
low expression levels could indicate (just a few thoughts):
- the protein is not folded properly and already degraded during
production which in turn may indicate problems with the construct
(insert + plasmid) itself or problems with the speed of expression (i.e.
no time to fold). Lower the expression temperature or induce at a much
higher OD600 (near log phase) and grow for 2 hours only after induction
- it is not always advisable to keep and purify the protein of interest
at 4ºCelsius (keyword "cold denaturation")
Things to try:
- implement the method described in the paper: Optimum solubility (OS)
screening: an efficient method to optimize buffer conditions for
homogeneity and crystallization of proteins, ACTA D D60, 1670-1673
(Jancarik et al.)
-At pH 7.5 and pI 8.6, your protein will be positively charged. Trying
to "stabilize" the protein according to the Hofmeister series of salts
is not helpful. Remember, the Hofmeister series of salts will inverse
with positively charged proteins! Meaning, NaCl is not a good salt to
use in order to solubilize the protein. Try to add 50-200 mM
ammoniumsulfate to help solubilize the protein.
Good luck,
Jeroen
Am 19.07.20 um 11:07 schrieb 张士军:
Dear all:
Any ideas to decrease protein polymer formation? my protein was easy
to form oligomers and precipitation when do purification,I have tried
add glycerol and DTT,both didn't work. Does anyone has experience to
avoid it happening. Thanks!
Best Regards
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*Dr.math. et dis. nat.Jeroen R. Mesters*
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