a tale of a transposase - p4eu · a tale of a transposase new purification protocol and...

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

2

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


• 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


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!


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!


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?


• 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


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


SEC with and w/o oligo Superdex75 10/300 GL, GE

- Oligo

+ Oligovoid

void

~100kDa

Tn5 dimer

~100kDa

Tn5 dimer

High A250

Oligos?


Thermofluor

Buffer Screen

RUBIC Buffer Screen

Molecular Dimensions

• Buffer substances

• pH values

• Salt concentrations


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


Final optimised Tn5 preparation

Samples to be tested:

• Pre-SEC sample

• 1st peak SEC


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


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


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


Plot by Lars Velten,

Steinmetz Lab, EMBL Heidelberg

Acknowledgements

Bianca Hennig

Lars Velten Steinmetz Group, EMBL

Chelsea Szu Tu

Paul Collier GeneCore EMBL

Vladimir Benes

Kim Remans PEPCore EMBL

Hüseyin Besir

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