In the beginning there was Jack
getting started here... The background reading I have done that will weigh heavy today:
The Pierce Refolding Kit Instructions[1] that detail the 9 buffer components in their kit, which are:
reducing agents
PEG
divalent cations
high [NaCl]
L-arginine
guanidinium chloride (GnCl)
and an unreleased "Refolding Guide" that comes with the ProMatrix kit an is not available electronically
A paper on High throughput automated refolding [2] that describes a 96 well matrix of buffer conditions they developed for structural genomics projects for Mtb and Viruses.
Finally, Vertex Pharmaceuticals [3] fills the gaps and describes the use statistical software to generate a fractional factorial screen; application of light absorption to measure protein precipitation (λ 390nm); and most importantly statistical analysis of the data (both precipitation and enzyme activity)from the fractional factorial matrix to draw conclusions and further direct optimization.
Designing Our Matrix
The problem with any matrix report, is that they are packed full of misc. chemicals that are either not in house, or prohibitively expensive... it is my experience to find a way to furnish results that are desired first, then ask for $$$ chemicals to improve future experiments second...
This excludes:
all Non Detergent SulfoBetaines (NDSB's) that can or cannot be important
a-cyclodextrin and methyl-B-cyclodextrin (NB: B-cyclodextrin that we have in house is not soluble in H2O.)
fancy detergents (lauryl maltoside)
N-lauryl sarcosine (iFOLD kits)
FoldACEs (iFOLD series 3 kit)
disco reducing agents (BMC: bis-mercaptoacetamide cyclohexane)
additionally,
From Willis' [3] report, buffer pH, detergent, and NDSB 201 were important for some proteins,
So, our matrix will include an expansion of pH, and various detergents around that may or may not help (it's a screen for a reason)
additives that are not NDSB's that may help, and some other stuff I like to hypothesize about.
The conditions we will screen follow:
pH/ Buffer
5.5 MES
6.5 MES
7.5 HEPES
8.2 TRIS
9.0 CHES
I replaced BORATE pH 9.5 from [3] with CHES to alleviate complications that will arise during reagent preparation, since borate is only marginally soluble in water, and cannot be prepared as a 10X stock (500 mM).
detergents/ concentration
triton X 100
tween 80
Chaps
none
NB: NONE is a category for all subsequent categories, because it must be included for matrix generation
reducing agents/ concentration
DTT
TCEP
β-ME
none
additives:
arginine
asparate
divalent cations (2 Mg++/ 0.1 Ca++)
GnCl
Ligand
PEG 3350
Glycerol
Sucrose
BSA
NaCl/KCl (high ionic strength)
Why BSA? it just works for me.
References
- [Pierce]
- Vincentelli R, Canaan S, Campanacci V, Valencia C, Maurin D, Frassinetti F, Scappucini-Calvo L, Bourne Y, Cambillau C, and Bignon C. High-throughput automated refolding screening of inclusion bodies. Protein Sci. 2004 Oct;13(10):2782-92. DOI:10.1110/ps.04806004 | PubMed ID:15388864 | HubMed [Vincentelli]
- Willis MS, Hogan JK, Prabhakar P, Liu X, Tsai K, Wei Y, and Fox T. Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions. Protein Sci. 2005 Jul;14(7):1818-26. DOI:10.1110/ps.051433205 | PubMed ID:15937284 | HubMed [Willis]
All Medline abstracts: PubMed | HubMed
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Refolding Matrix
