ProteoCool Pills#6: Rapid protein Expression and solubility check using a 24 tip-horn sonicator

 

The use of high cell density media (e.g Enpresso reported in the ProteoCool n°7 or YGR reported in the ProteoCool n°32 ) for protein expression using E.coli allows to scale down culture volumes and easily express many clones in parallel.

Protein expression and solubility check can become a limiting step and the set-up of a simple and reliable protocol is essential.

Automation by using robotic platform is a possible solution for a real high-throughput approach, (e.g a screening projects that require to process  thousands samples for week) but in the most cases just the implementation of a optimized and rational protocol based on the use of multi-channel pipettes and 96-deep well plates can allow to manipulate in parallel up to hundreds samples/week with low cost and high protocol flexibility.

The lysis method used in the small scale test need to be similar to the ones that will be applied for the purification of the protein from rest of biomass in case of positive results, and therefore Sonication or is preferable respect than freeze-thaw cycles (work well in small scale but is not scalable) and chemical/enzymatic lysis ( expensive in scale-up)

Protocool

1) About 8OD of bacterial cells (for Enpresso correspond to 800ul not induced or 500ul induced 8h or 300 ul induced 24h) were transferred from culture into the 96 Deepwell plate following the scheme reported below (to fit the 24 tip sonicator horm)

2) Plate were centrifuged for 20' at 3700 rpm and 4°C

3) After centrifugation the surnatants were discharged 

4)    The pellets were resuspended adding 960ul of cell Lysis/binding buffer ( eg: Tris 20mM, NaCl 300mM, imidazole 10mM pH=8 for His-tagged protein that will be purified by IMAC) and mixed 3-5 times with the multichannel to allow complete pellet re-suspension. 

2)    Cell Lysis by sonication using the 24-tip horn:

Sonication cicle: 30’’ ON/45’’ OFF , 20 cycles  50% power – Qsonica Q700 sonicator.

The plate need be placed in ICE during the sonication to avoid sample heating.

3)    From each well 60ul of sonicated solution (Total fraction) e transferred in PCR plate no skirted containing 20ul of Loading sample buffer 4X ( eg. Thermo scientific cod. NP0008 ) with reducing agent.

4)    The 96-deepwell plate were centrifuged at 20’ a 4000rpm 4°C;

5)    From each well 60ul of surnatant (Souble fraction) e transferred in PCR plate no skirted containing 20ul of Loading sample buffer 4X ( eg. Thermo scientific cod. NP0008 ) with reducing agent.

6)    The no skirted PCR plate is closed by aluminum foil and incubated 5’ a 95°C in a PCR machine.

7)   12ul of each samples were loaded in a 26well 4-12% SDS-page gel.

Example of results: