a tale of a transposase - p4eu · a tale of a transposase new purification protocol and...
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A tale of a Transposase
New purification protocol and
optimisation of long term storage
conditions of Tn5 Transposase
Ines Racké
EMBL Heidelberg
Tagmentation based library construction
Figures by Bianca Hennig
Steinmetz Lab, EMBL Heidelberg
P4EU Meeting June 2016 EMBL Heidelberg1
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Tn5 Transposase
• Commercially available from Illumina (Nextera®)
• The goal is to start with “homemade” Tn5 transposase
and Nextera® reagents and then stepwise replace all
reagents from kit to be completely independent from
Illumina
P4EU Meeting June 2016 EMBL Heidelberg
http://www.ebi.ac.uk/
• Construct in pTXB1 vector (NEB), allele for hyperactive Tn5 from
Esteban Martinez-Garcia (Martinez-Garcia et al. 2011)
• wt Tn5 has very low activity 2 mutations to make it
“hyperactive”
• Expression in C3013 cells (NEB)
• Intein-CBD fusion protein
Expression of hyperactive Tn5
Picelli et al.; Genome Res., July 30, 2014
P4EU Meeting June 2016 EMBL Heidelberg3
• Lysis buffer: 20mM HEPES-NaOH pH 7.2, 800mM NaCl, 20mM Imidazole, 1mM EDTA, 2mM DTT, 10% glycerol, 0.2% Triton X-100
• PEI precipitation step
• Elution with 100mM DTT
• Yield ~15mg fusion protein
from 1L expr culture
Purification of hyperactive Tn5
P4EU Meeting June 2016 EMBL Heidelberg4
Picelli et al.; Genome Res., July 30, 2014
November 2014
• Construct, E. coli strain and protocol from Picelli paper
• Chitin resin in batch instead of packed column on FPLC
https://openi.nlm.nih.gov/
Yield was very low!
P4EU Meeting June 2016 EMBL Heidelberg5
February 2015
• We suggested to clone into pETM11-SUMO3
• Steinmetz group did the cloning
• Same Tn5 allele as in Picelli paper: classical E54K and L372P
mutations, wild type M56
• N-term HisSUMO3 fusion tag SenP2 cleavage
BL21 (DE3) CodonPlus RIL
Best condition ON 18ºC 0.2mM IPTG
High overexpression, very promising!
P4EU Meeting June 2016 EMBL Heidelberg6
HisSUMO3 Tn5 purification
• Simple NiNTA purification on AEKTA Purifier
• PEI step and buffer conditions from Picelli paper
• Yield ~10mg cleaved Tn5 from 1L culture
Better yield, active protein, but how to store it?
P4EU Meeting June 2016 EMBL Heidelberg7
• Lysis in Standard buffer containing Triton X-100
May 2015 Tn5-SUMO3 test purification with and w/o Triton X-100
Both were stored w/o Triton:
20mM HEPES-NaOH, pH7.2
800mM NaCl
1mM EDTA
2mM DTT
10% glycerol
Previous storage buffer:
20mM HEPES-NaOH, pH7.2
100mM NaCl
1mM EDTA
2mM DTT
10% glycerol
0.2% Triton X-100
Samples w/o Triton are better
Activity comparable to Nextera and reproducible
Only active for ~3 months storage
Purification with Triton X-100 in all buffers Purification without Triton X-100
P4EU Meeting June 2016 EMBL Heidelberg8
December 2015
• Lysis in high salt and with Triton X-100
• Purification buffers w/o Triton X-100
• Low salt in IEX is bad for Tn5 → 3rd step SEC instead of
IEX
• Comparison SEC with and without Oligo attached to Tn5
• Thermofluor
P4EU Meeting June 2016 EMBL Heidelberg9
SEC with and w/o oligo Superdex75 10/300 GL, GE
- Oligo
+ Oligovoid
void
~100kDa
Tn5 dimer
~100kDa
Tn5 dimer
High A250
Oligos?
P4EU Meeting June 2016 EMBL Heidelberg10
Thermofluor
Buffer Screen
RUBIC Buffer Screen
Molecular Dimensions
• Buffer substances
• pH values
• Salt concentrations
P4EU Meeting June 2016 EMBL Heidelberg11
Additives Screen
RUBIC Additive Screen MD1-97
Molecular Dimensions
• Nucleotides
• Aminoacids
• Metal ions
• Sugars
• Detergents
Melting temperature ~38,5ºC
No condition could stabilise Tn5 significantly
February 2016
• Final attempt including all changes and improvements
• Comparison of long term storage conditions
• Lysis with high salt and Triton X-100, purification buffers w/o
• 3rd step SEC
• Storage buffer with 800mM NaCl, 50% glycerol and w/o Triton X-100
• Prep from 1L yield before SEC 70mg Tn5
P4EU Meeting June 2016 EMBL Heidelberg12
Final optimised Tn5 preparation
Samples to be tested:
• Pre-SEC sample
• 1st peak SEC
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Final sample (Pooled fractions from
2nd peaks from 2 SEC runs, 50%
glycerol)
Final optimised Tn5 preparation
• Final sample was re-run on SEC to check whether there is an
equilibrium between the dimer and the oligomer or whether dimer is
stable
• “Dimer peak” is bigger than “aggregate peak” suggesting that
Dimer is relatively stable
P4EU Meeting June 2016 EMBL Heidelberg14
Storage conditions
• Storage buffer 50mM Tris-HCl (pH 7.5), 800mM NaCl, 0.1mM EDTA,
1mM DTT, 50% glycerol
• Half of the aliquots: Stored @ -20ºC without shock freezing
• Half of the aliquots: Stored @ -80ºC after shock freezing in liquid N2
• Storage w/o oligo
P4EU Meeting June 2016 EMBL Heidelberg15
And they all lived happily ever after…
• Long term storage problem solved
• All Nextera reagents replaced by
homemade or cheaper commercial
components
• Homemade Tn5 + components <20ct per library
Illumina 8€ per library
• > 2000 libraries constructed with our homemade Tn5
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Plot by Lars Velten,
Steinmetz Lab, EMBL Heidelberg