Can you try with trizole or thaw the sample and uses the Qiagen RNA extraction Minikit.

You could use 293T cells, pool the plates, and the purify. Production is the easy part; you need someone skilled with gradient columns and affinity chromatography to have your protein pure enough to get its atomic structure.

And if you decide to use a baculovirus expression system, here is the reference of a paper in which we reported the crystal structure of BTK produceduced in insect ovary cells: www.jbc.org/content/276/44/41435.abstract

Hi,

For crystallization purpose, its good to express your protein in bacteria as you could get mass scale protein and would require to purify your protein as in get specific protein bands in an SDS gel. You might need to play around with conc. of salts for the purification purpose and also take care whether your protein come out in the supernatant fraction during centrifugation or are in pellets or inclusion bodies. It all depends on the kind of vector used for expression and property of protein. You would go for IPTG induction.

While of you would go for higher expression using cell lines, as mentioned HEK 293T is really good as the transfection efficiency is atleast 90% and you would need to scale your cultures up. may be T75 flasks, pool and quantify. I would suggest that when you are transfecting T75 say three in no., make a common DNA-reagent mix and then from the same mix add it to individual flasks to reduce variability in expression.

Hope the link helps.

http://web.mnstate.edu/provost/Recombexpresguide.pdf

Thanks for all your suggestion and i will go through the reference article which u referred.. Then i will come back with doubts ..........

Thank you so much

Expressing in 293T will be too expensive for crystallization. If you know the cloning sites, try digesting the construct with the respective enzymes and recloning the insert to a bacterial expression vector, say pET or pMAL. That would make life easier. All the best.

Thank you Mayilaadumveettil Nishana. i am also planning to like that only........

Hi Kannan:

Since your GOI is already in pCDNA3.1, you may consider transient transfection using the 293FS system. Is your target a cytoplasmic, membrane or secreted protein?

I would also suggest that you try small scale expression in various cell lines before moving onto a large scale system for crystallization. In any event, here are a few references that might help-

1) Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM (2008) Rational vector design and multipathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection

under serum-free conditions. Nucleic Acids Res 36:e96

2) Hopkins RF, Wall VE, Esposito D (2012) Optimizing transient recombinant protein expression in mammalian cells. In: Hartley JL (ed) Protein expression in mammalian cells, vol 801, Methods in molecular biology. Humana Press, NY pp 251–268

3) Geisse S, Fux C (2009) Recombinant protein production by transient gene transfer into mammalian cells. In: Richard RB, Murray PD (eds) Methods in

Enzymology, vol 463. Academic Press, pp 223–238

Good Luck.

Mallika

Thanks Mallika Sastry