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)
also contained in the kit is an unreleased "Refolding Guide" that comes with the ProMatrix kit an is not available electronically... it would be nice to read this, but C'est la vie!
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 report on buffer matricies is that they are packed full of miscellaneous chemicals that are either not in house, or prohibitively expensive... it is my experience (professor management theory) that the best way to get expensive materials/equpment to support a new method/tehcnique is to furnish results that are desired but were only accessible from that new method/technique (grunt work), and then ask for $$$ chemicals to improve future experiments...
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
9.2 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
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
DTT
TCEP
none
additives:
arginine/asparate/none
divalent cations (2 Mg++/ 0.1 Ca++)
GnCl
Ligand
PEG 3350/Glycerol/Sucrose
BSA
NaCl/KCl (high ionic strength)
Every additive looks reasonable except for 1... BSA.
And, why BSA? this is my screen, and a I want to try something wacky.
Stock Solutions
it is important to make sure that your stocks are at concentrations appropriate such that an all positive sample (e.g. one having all possible components) does not have a volume greater than the total volume for the experiment...and that all components are soluble at the concentration determined here for the stock solution.
here is the table I cooked up to figure out what "X" concentration I would need to make everything work, taking into account certain characteristics (e.g. if 20% glycerol will be the final concentration, then i cannot achieve more than a 5X stock solution)
| component
|
stock solution
|
relative volume
|
| Buffer |
10X |
10
|
| detergent |
100X |
1
|
| amino acids |
5X |
20
|
| GnCl |
10X |
10
|
| Ligand |
10X |
10
|
| PEG |
100X |
1
|
| Glycerol |
5X |
20
|
| Salt MIX |
10X |
10
|
| BSA |
10X |
10
|
| Reducing agent |
100X |
1
|
|
total |
93
|
|
|
NB: I used the totally wicked excel2wiki converter for the table code
so we are set.
| component
|
concentrations from [3]
|
units
|
interconversion
|
| Buffer |
50 |
mM |
|
| detergent |
0.5 |
mM |
~0.06%
|
| amino acids |
550 |
mM |
|
| GnCl |
550 |
mM |
|
| Ligand |
100 |
uM |
|
| PEG |
0.06 |
% |
|
| Glycerol |
20 |
% |
|
| Salt MIX |
264/11 |
mM Na/K |
|
| BSA |
10 |
mg/mL |
|
| Reducing agent |
5 |
mM |
|
| Divalent cation |
2 |
mM |
|
|
|
stock solutions we will need:
| stock solution
|
concentration
|
units
|
| Buffer |
500 |
mM
|
| detergent |
6 |
%
|
| amino acids |
5500 |
mM
|
| GnCl |
5500 |
mM
|
| Ligand |
1000 |
uM
|
| PEG |
6 |
%
|
| Glycerol |
100 |
%
|
| Salt MIX |
2640/110 |
mM Na/K
|
| BSA |
100 |
mg/mL
|
| Reducing agent |
500 |
mM
|
| Divalent cation |
200 |
mM
|
|
|
Buffers
| Buffer
|
molecular weight
|
stock solution
|
volume
|
mass
|
'
|
| MES |
195 |
500 |
50 |
4.875 |
g
|
| HEPES |
238.3 |
500 |
50 |
5.9575 |
g
|
| TRIS |
121.1 |
500 |
50 |
3.0275 |
g
|
| CHES |
207.3 |
500 |
50 |
5.1825 |
g
|
|
|
salt mix
| high salt
|
component
|
stock conc
|
units
|
desired conc
|
vol
|
units
|
volumes
|
'
|
|
NaCl |
5 |
M |
2.64 |
50 |
mL |
26.4 |
mL
|
|
KCl |
4 |
M |
0.11 |
50 |
mL |
1.375 |
mL
|
|
water |
|
|
|
|
|
22.225 |
mL
|
| low salt |
|
|
|
|
|
|
|
|
|
NaCl |
5 |
M |
0.264 |
50 |
mL |
2.64 |
mL
|
|
KCl |
4 |
M |
0.011 |
50 |
mL |
0.1375 |
mL
|
|
water |
|
|
|
|
|
47.2225 |
mL
|
|
|
divalent cation mix
| divalent cations
|
'
|
'
|
stock
|
'
|
vol tot
|
'
|
vol
|
'
|
| MgCl2 |
200 |
mM |
1 |
M |
25 |
mL |
5 |
mL
|
| CaCl2 |
10 |
mM |
1 |
M |
25 |
mL |
0.25 |
mL
|
|
|
|
|
|
|
total |
5.25 |
mL
|
|
|
|
|
|
|
|
|
vol water |
19.75 |
mL
|
|
|
detergents
| detergents
|
stock
|
|
desired concentration
|
|
|
|
stock (mL)
|
water (mL)
|
| TritonX |
25 |
% |
6 |
% |
25 |
mL |
6 |
19
|
| Tween 80 |
20 |
% |
6 |
% |
25 |
mL |
7.5 |
17.5
|
| CHAPS |
|
|
6 |
% |
25 |
mL |
1.5 g |
to 25
|
|
|
ligand
| ligand
|
stock
|
'
|
desired
|
'
|
vol
|
'
|
stock (uL)
|
'
|
water
|
'
|
| Ligand A |
20 |
mM |
1 |
mM |
10 |
mL |
500 |
uL |
9.5 |
mL
|
|
|
amino acids
| amino acid
|
mw
|
'
|
stock
|
'
|
vol
|
'
|
mass
|
| L-glutamate |
147 |
g/mol |
5.5 |
M |
50 |
mL |
40.425
|
| L-arginine |
211 |
g/mol |
5.5 |
M |
50 |
mL |
58.025
|
|
|
reducing agents
NB: stocks will be prepared without reducing agents! these should be added fresh every time
| reducing agents
|
mw
|
'
|
stock
|
'
|
vol
|
'
|
mass
|
'
|
| DTT |
155 |
g/mol |
0.5 |
M |
10 |
mL |
0.775 |
g
|
| TCEP |
250 |
g/mol |
0.5 |
M |
10 |
mL |
1.25 |
g
|
|
|
Matrix
the first matrix can be found here
The above conditions were paired in "Custom Design" of JMP-IN as follows:
pH: 5 level Categorical
Detergent: 4 categories (none, triton, tween, chaps)
amino acids: 3 categories (none, arg, glu)
salts: none low high
crowders: none PEG Glycerol
divalent cation: 0, 1
GnCl: : 0, 1
BSA: 0, 1
Ligand: 0, 1
Reducing agent: 0, 1
the second matrix matrix can be found here
or the following matrix with the amino acids as a 4-factor categorical with (none, glu, arg, glu+arg)
I am partial to exploring the largest number of possible buffer configurations, but I want it to still be worth while, i.e. if one of these randomized ingredients has negative effects on folding, a large portion of the wells is worthless.
it took all afternoon to prep the stock solutions above. Neither pH meter probe works, and we do not have any solid TCEP.
I will go to the Core tomorrow and acquire some fresh DTT and TCEP.
NOTE: both amino acid solutions were adjusted to be 1/2 the concentration here. 58g Arg in 50 mL water (116% w/v solution) seemed a bit over the top.
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
|
Refolding Matrix
