ANÁLISIS DEL CONSUMIDOR

The rCdc6 protein was produced with a 6 his tag at the N terminal. This tag is

designed to facilitate binding to transition metal cations and Nickel-agarose (Ni-NTA

agarose) (Qiagen) was chosen for affinity purification. The NT A group occupies four of

the ligand binding sites around the nickel ion, leaving two sites for the stable interaction

by 6xHis tagged proteins. To reduce the binding of contaminants to Ni-NTA a low

includes an imidizole ring and it is this ring which binds Ni-NTA. Imidizole itself has an

affinity for Ni-NTA and at low concentrations it can compete with and prevent the

binding of low affinity contaminants. A high concentration (200mM), of Imidazole can

compete with 6xHis tagged proteins for binding of Ni-NTA and these conditions can be

used to elute proteins from the resin.

Previous work in our laboratory has shown that Cdc6p precipitates at low ionic

strengths and thus, requires high salt conditions to remain soluble during purification

(Gordon Perkins personnel communication). Since Cdc6p is soluble in cell extracts

which contain a final salt concentration equivalent to 600mM potassium glutamate, I

decided to use these conditions for the purification of rCdc6p. The buffer used for each

step of the purification includes 600mM potassium glutamate. Other components are

listed below.

CDC6 expression was induced in the FB810 pET15b-CZ)Cd and BL21-Codon Plus™ pET15b-CDCd strains using IPTG, as described previously. The cell pellets from

the FB810 strain were frozen in liquid nitrogen then resuspended in KGI20 (5mM (3-

mercaptoethanol, 20mM imidazole, 40mM hepes-KOH pH 7.8, 160mM sorbitol, 0.1%

triton xlOO, 2mM magnesium acetate, 0.25mM AEBSF, 600mM potassium glutamate).

Resuspension caused a large proportion of cells to lyse and this process was then

completed by sonication. The cell pellets from the Codon+ strain had to be treated more

harshly to induce lysis and this included washes with an EDTA buffer, digestion with

lysozyme and strong sonication, and is described in detail in section 2.6.3. The Codon+

To test the solubility of rCdc6 in the cell extract from the Codon+ strain, it was

centrifuged at 30,000xg for 15 minutes (4°C) and samples taken before and after

centrifugation. Both cell extracts were then loaded onto Ni-NTA agarose columns

(described in section 2.6.5) and samples taken and analysed by immunoblotting. Figure

14a shows samples from the FB810 pET15b-CDCd column and Figure 14b samples from

the BL21-Codon Plus™ pET15b-CDCd column. In both cases 4|xl of extract (from

80ml), 4p.l of flow through (from 80ml), 8p.l of wash (from 25ml) and 8p,l of each

fraction (from 1ml fractions) were analysed by immunoblotting with a monoclonal raised

against Cdc6p.

A small proportion of the rCdc6p from the FB810 strain (A) was detected in the

flow through of the Ni-NTA agarose column. There was also a small amount of rCdc6p

detected in the wash, however most rCdc6p bound to the Ni-NTA agarose resin and was

eluted during the imidazole gradient.

The rCdc6p from the Codon+ strain (B) could not be purified using the conditions

described. This protein did not precipitate as it was present in the whole cell extract after

centrifugation, however it was also detected at high levels in the flow through of the

column. Because no rCdc6p was detected in the fractions of the imidazole gradient,

rCdc6p could not associate with Ni-NTA under these conditions, and thus, flowed

through the column.

The difference between the rCdc6 proteins from the different extracts could be

due to different conditions in the cells o f the two strains, the difference in the

Figure 14

Ni-NTA chromatography of rCdc6p from FB810 and Codon

Plus™ pET15b-CZ)C6 strains.

1 litre of FB810 pET15b-CZ)C5 (A) and BL21-Codon Plus"" pET15b-CDCd (B) were grown and rCdc6p with a 6xHis-tag was induced as described in Figure 12. Both strains were lysed (see 2.6.3), the conditions for the Codon+ strain lysis being most harsh, as these cells do not produce T7 lysozyme. Cell extracts were centrifuged to remove insoluble protein and other cell debris, then loaded onto 7ml Ni-NTA agarose columns in KGI20 buffer (5mM (3-mercaptoethanol, 20mM imidazole, 40mM hepes-KOH pH 7.8, 160mM sorbitol, 0.1% triton xlOO, 2mM magnesium acetate, 0.25mM AEBSF, 600mM potassium glutamate) and the flow through collected. Columns were washed with 3.5 column volumes of KGI30 then proteins eluted with a gradient of imidazole from KGI30 to KGI200 (200mM Imidazole).

Samples were taken from each stage o f the protocols and analysed by immunoblotting for Cdc6p. 4pil (from 80ml) of the Charge, 4|nl (from 80ml) o f Flow through, 8p,l (from 25ml) of Wash and 8p,l of the 1ml fractions were analysed.

14A 9^ Elution fractions q \ ^ ^

% %

I decided to investigate whether the behaviour of rCdc6p during Ni-NTA agarose

chromatography could be influenced by the ionic conditions chosen for purification, and

the results are described in the next section.

5.4 The ionic conditions for Ni-NTA agarose purification