Crystals with Organic solvents

From: eswar reddy
Date: 26 August 2011 11:56

Dear All 

I was working on a Human protein and expression and solubility is good in E.coli  and purification is One step (His-Tag), and i need to cleave the Histag before screens, if not the protein will precipitated and Aggregated, but after trying for 1.2 years i have crystals and they are with Organic solvents, (10 conditions), these crystals are inter grown like broccoli  shaped  and i tried seeding, but it is not successful, and even i tried  with additive screen but the result is the same .... is there is any idea to increase the size and shape of my protein crystals.

Any suggestions will be helpful for me 

Thanks in Advance   

----------
From: Bosch, Juergen

Depending on how big your broccoli is I would simply crush them and mount as big of a piece as possible and see how well it diffracts.

I assume you have tried already:

- glycerol

- changing protein:reservoir ratios

- temperature

- adding oil to either / and the reservoir, your drop

-crushed up some broccoli and recreened your original sparse matrix screen while seeding with chunks of broccoli seeds 

- when you say the His-tag needs to be cleaved otherwise it will precipitate, that mitt also just be either leaching Ni from your column or simply the imidazole

- You don't say how you purify your protein, I hope you are not just setting up after the Ni capture step and at least have some other method following e.g. anion or SEC

Just a few incomplete suggestions,

Jürgen

......................

Jürgen Bosch

----------
From: Van Den Berg, Bert
I would definitely try gelfiltration (how do you get rid of the cleaved tag anyway, sample buffer exchange?) but especially ion exchange. A homogeneous sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in ion exchange. Beyond that i would make some point mutations on surface residues and focus on those (ie try the surface entropy method).
Good luck, Bert

----------
From: Patrick Shaw Stewart
Hi Eswar

Firstly, I would certainly try crystal seeding into random screens if
you haven't already tried it.  Refs below.

Secondly, it's very convenient to grow the crystals under oil, and to
soak the organic solvents into the drops, through the oil.  This makes
it much easier to harvest the crystals because the oil becomes
saturated with the solvent and stops it from evaporating when you pick
up the crystals. This approach can be used at the screening stage too.

See Mortuza, et al. High-resolution structure of a retroviral capsid
hexameric amino-terminal domain.  Nature 431 (2004), pp 481-485.  Also
see http://www.douglas.co.uk/winner1.htm

Good luck

Patrick

________

Allan D'Arcy, Frederic Villarda, May Marsh.  'An automated microseed
matrix-screening method for protein crystallization'.  Acta
Crystallographica section D63 (2007) 550–554.  On-line at
http://scripts.iucr.org/cgi-bin/paper?S0907444907007652

A. G. Villaseñor, A. Wong, A. Shao, A. Garg, A. Kuglstatter and S. F.
Harris. 'Acoustic matrix microseeding: improving protein crystal
growth with minimal chemical bias.' Acta Crystallographica Section D66
(2010) 568-576. On-line at
http://scripts.iucr.org/cgi-bin/paper?S0907444910005512

Galina Obmolova,* Thomas J. Malia, Alexey Teplyakov, Raymond Sweet and
Gary L. Gilliland. 'Promoting crystallization of antibody–antigen
complexes via microseed matrix screening.' Acta Crystallographica
Section D66 (2010) 927–933.  Open-access at
http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf

Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E.
Chayen and Peter F.M. Baldock. 'Getting the most out of the random
microseed matrix-screening method in protein crystallization'.  Cryst.
Growth Des., 2011, 11 (8), pp 3432–3441. On-line at
http://pubs.acs.org/doi/abs/10.1021/cg2001442

See also http://www.douglas.co.uk/mms.htm

--
 patrick at douglas.co.uk    Douglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090    US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36