(by cathalgarvey from gmail.com)
Sat Sep 25 04:18:03 EST 2010
Is it possible that the testis has a different tRNA use profile, which might
be woefully inappropriate in normal tissue or E.coli?
Could any microRNAs be interfering in normal tissues?
Sent from my beloved Android phone.
On 24 Sep 2010 22:04, "Peter Ellis" <pjie2 from cam.ac.uk> wrote:
The protein I'm working on doesn't want to express in any culture or in
vitro system I've tried. So far I've been through:
pEXT5-CT vector (T7 promoter, C-terminal His tag). No soluble or insoluble
expression in BL21DE3 cells with autoinduction or with IPTG induction. No
soluble or insoluble expression using coupled T7 transcription/translation
systems, either with E. coli extract or reticulocyte lysate.
pT7CFE1 vector (T7 promoter, C terminal His tag, ribosome entry site and
3'UTR optimised for use in vitro transcription/translation with mammalian
extracts). No soluble or insoluble expression with reticulocyte lysate
pGEX(KG) vector (tac promoter, N terminal GST fusion). No soluble or
insoluble expression using autoinduction or IPTG induction.
pEGFP-N1 vector (CMV promoter, N terminal GFP fusion). No soluble or
insoluble expression in two different mammalian cell lines.
All four of the above systems have worked fine in our hands with control
genes (and some other genes I'm working on), so it's nothing wrong with the
kits/cells or our lab technique. For the latest attempt at in vitro
transcription/translation, we isolated the mRNA from the reaction and
sequenced it, so we know the constructs are being expressed and is in-frame
etc. The protein just doesn't want to translate, no matter what system we
I guess my protein of interest just really doesn't like being expressed in
culture! Maybe it needs particular cofactors / chaperones in order to be
translated? Does anyone have any suggestions what to try next? The protein
itself is about 50kDa, specific to round spermatids in the testis.
Spermatids are pretty weird cells, so it's certainly possible that my
protein isn't happy in any other cell type. Unfortunately round spermatids
don't grow in culture, so transfecting spermatids directly isn't an option.
About all I can think to try is to add some testis extract to an in vitro
transcription/translation reaction and hope it supplies whatever the missing
factors are. Has anyone tried anything like that before, and is it a
remotely sensible thing to try? If so, what's the best way to go about it -
homogenise testis tissue in RIPA buffer or PBS and just add some to the
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