Hello Protein gurus!
I’m trying to express a human 55 kDa protein using a GST fusion
approach, vector is
pGEX4T3. I switched to GST (from His-Tag) since the lab down the hall
had GST
going well, a tried and sometimes true rationale in science!
Cells are grown and induced for 3 hours, then lysozyme and sonicated,
lysates are put
on GST-sepharose and eluted. For quick looks sometimes I just look at
lysates.
Culture volumes are 1 ml, of which 25% of the lysate or GST-sepharose
eluate is put
in a single mini-protein gel lane. When using the GST-sepharose I grow
10 ml
cultures.
In all cases the vector alone gives lovely bands by immunoblotting, so I
know my
semi-dry blotting and anti-GST and secondary Ab is working!
There are no fusion bands around 82 kDa, only a weak band that
corresponds to GST
vector alone.
Conditions tested:
E.coli BL21 Codon strain (Stratagene), E.coli Top 10 (Invitrogen)
+/- 1 mM IPTG (three hour induction)
+/- 1 % glucose
growth at 28C vs 37C (with 1% glucose)
Native (sonicated) vs Denaturing (Urea)
When doing a larger prep using GST-sepharose, I’ve even tried binding
the putative
protein out of the media, the lysate, the insoluble pellet, no luck.
Before you ask:
Clone is "in frame" relative to the gst.
No stop codon is introduced between gst and the ATG of the gene
The native stop codons are preserved in frame.
Protease Inhibitors: PMSF, benzamidine, and Inhibitor (EDTA-free)
cocktail tablets.
Previously I was trying to work with His-Tags constructs. The first
approach was a
amino-terminal His-tag. After a couple months of amibigious results and
talking to
people at meetings I switched the His Tag to the carboxy terminal end.
It turns out
that frameshift mutations occur in the coding sequence gene either due
to the Taq or
possibly by forcing a mutation to deactivate a “toxic” protein. The
sequence of the
cloned PCR product prior to cloning is OK, but only a couple of colonies
arose when
cloning into the pTrcHis vector. This is when I switched to GST, and
used Pfu to
amplify out of cDNA. Overall I am more impressed with the ability of
GST-Sepharose to bind to controls than His-Tag.
I have not varied the length of induction, nor the concentration of
IPTG. Can anyone
out there offer any suggestions as to which conditions to vary. Is this
doomed to fail?
Should I start looking at baculovirus or gateway technology?
Thanks for listening to this drawn out, painful experience!
Frank
rozsa at umich.edu
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<( @ @ )>
------oOOo--(..)--oOOo-------
Frank W. Rozsa, MPH, PhD
Research Investigator: Glaucoma Genetics
Dept. of Ophthalmology
Kellogg Eye Center
University of Michigan
1000 Wall Street
Ann Arbor, MI 48105
email: rozsa at umich.edu
alt: cmotdibbler at hotmail.com\
alt2: frozsa at mac.com
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