try codon optimisation
use Rosetta 2 strains
fuse to MBP
I expressed a yeast protein of ~100 KDa, expression was low until I fused it
to MBp (resulting protein ~ 150 KDa)
Hi,
I would like to add one thing to the already good list of suggestions.
You can try to express your protein in Lactococcus lactis. We've
earlier had successful expression in lactis where it was none or only
inclusion bodies in E. coli. See for example: Kunji et al. Biochim
Biophys Act
May be this could help,
*Protein Expression and Purification 45 (2006) 191–199*
* In vitro activation, purification, and characterization
of Escherichia coli expressed aryl-alcohol oxidase,
a unique H2O2-producing enzyme.
Francisco J.
With the disclaimer that I have yet to work with a flavoprotein, is there
enough FAD around in your culture to allow for proper folding of your
protein ? Maybe adding riboflavin to you media, or trying ultimately to
refold you protein from inclusion bodies (which will be very pure) in the
presence
e 29, 2009 4:55 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] How to express a >95KD FAD protein
Dear all,
I know that there are a lot of experts having experience in expressing a big
protein in E.coli or yeast. My project is about a 95kd
covalently-FAD-binding protein (Human protein). I tr
a couple of quick things. This sounds like more of a protein-folding
issue. The lack of expression is probably caused by the cells clearing
of aggregated protein. You want to have conditions where the protein can
properly fold.
1) have you considered periplasmic expression by adding a periplas
Hi Wei Yong,
Sounds like a tricky situation.
A couple of things come to mind:
1) Have you tried expression from a synthetic gene? Sometimes the
mRNA is unstable and improving mRNA stability through optimization
(synthetic gene) helps.
2) Are you able to look at either human isoforms or ortho
Dear all,
I know that there are a lot of experts having experience in expressing a big
protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding
protein (Human protein). I tried very hard but have a bad luck over the past
1.5 years. I list what I did briefly so far. I am loo
