The Expi293 (see ProteoCool n° ) and Expi-CHO Expression System (Thermo) provides high-yield recombinant protein production in mammalian cells by combining high-density an optimized chemically defined media and transfection protocol.
Generally 'i'm performing the small scale expression and solubility trials in 2ml scale culture (in 6 well plates)
The availability of a simple IMAC Small scale purification protocol is essential in the preliminary expression tests to detect those proteins that are poorly expressed and/or co-localize with other proteins secreted in the cell surnatant.
Here I report a simple and rapid protocol that can allow to you to perform small IMAC purification for up to 24 sample in parallel in short time (1-2h):
1.
The cells were Transferred in a 2ml Eppendorf or 24 deep well plates
(depend from the number of sample tested);
2. Centrifugation at 300g for 5;
a) Cell Pellet: resuspended in 2ml of binding buffer, diluted 1/3 and loaded in SDS page;
b) Surnatant à go to step 3;
3.Centrifugation 10’ at maximum speed to completely remove cell debris (12000g in Eppendorf 2ml0
a) Pellet discharged;
b) Surnatant were diluted 1:1 with the IMAC binding
buffer (Tris 20mM, Nacl300mM, imidazole 10mM pH=8) and loaded
in the purification column* (go to step5);
* Buffer exchange is not necessary to obtain good
binding to the IMAC coloumn;
4.
Assembly of the purification column:
- Biorad Bio-Spin (cod.7326008) loaded with 100ul of Ni-Sepharose FF (Ni-Sepharose FF - Cytiva cod.17531801*)
*The same protocol was also performed using Ni- Sepharose Excel resin (GE cod. E17371201) those theoretically was developer for purification of protein from the mammalian cell suratant but in our hands the standard Ni-sepharose seems to provide best results in terms of protein purity;
-
Column was washed (for gravity)
with 2 ml of milliQ
water to remove the ethanol.
-
Column was equilibrated with 2ml of IMAC Binding
buffer;
Using a simple 4 position tube rack (VWR cod. 525-0951) and a large volume adjustable spacer multipipette (e.g RAININ LA6-1200XLS) up to 32 protein sample can be easily loaded (step 5) and washed in parallel (step 6)
5.
Protein sample were loaded
into the equilibrated column and F.T discharged.
6.
Columns were washed with 6ml of IMAC binding
buffer and buffer
discharged
7.
Elution was performed
with 300ul (3CV) of elution buffer (Tris 20mM, NaCL 300mM, imidazole 300mM pH=8) and the eluted fraction
is collected in a 2 ml
Eppendorf tube.
8.
To collect all the elute columns
were centrifuged 2’ at 3000rpm.
Example of result
obtained with this protocol:
where:
C- are non
transfected cells
C+ are Expi293 transfected with a 10His-tagged protein cloned in pcdna3.4 with an N_terminal leader sequence for secretion in mammalian cells.
The
culture volumes and the column size to be used in the scale up need to be
carefully select on the basis of
the expression level observed in the small-scale trials and of the amount of
protein that we need to produce.
Eg:
<0,5mg of protein: 100 of Ni-sepharose resin in a Biorad Bio-Spin coloumn
0,5-1mg of protein: 200-300ul Ni-sepharose resin in a Biorad Poliprep (cod. #7311550) Gravity flow ;
1-10 mg of protein:1-2ml di of Ni-Sepharose resin in a empty PD-10 gravity flow (a peristaltic pump could be used to perform sample loading, wash and elution)
>10mg of protein prepacked 5ml His-trap FF Crude connected to a peristaltic pump or AKTA purification system
Differently from the expression test, when we would like
to purify protein, all the fractions need to be collected and loaded in the SDS-page to follow the protein and
washing steps with buffers contain intermediate
imidazole concentration (e.g. 20mM for 6xHis-tagged protein or 30mM for
10xHis tagged proteins) are included in order to improve final protein purity.
The columns if correctly stored (must not go DRY and stored at 4°C in water or water/ethanol 20% for long time) are reusable for several time. Metal stripping and regeneration could be performed as already shown in the ProteoCool n° 25: Simple IMAC regeneration
