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? --- Twitter: @onetruecathal Sent from my beloved Android phone. On 24 Sep 2010 22:04, "Peter Ellis" <pj...@cam.ac.uk> wrote: Hiya, 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 transcription/translation system. 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 use. 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 reaction? Peter Ellis _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
