ANÁLISIS DE LA COMPETENCIA

To establish whether the ionic conditions chosen for purification of rCdc6 from

the BL21-Codon Plus™ pET15b-CDC(5 strain could affect the affinity of rCdc6 for Ni-

NTA agarose, I decided to perform small scale purifications in the presence of different

ions. As described in sections 2.6.4 and 2.6.5 four conditions were chosen. For the first

sample all chromatographic steps were performed in the presence o f 600mM potassium

glutamate. For the second sample all steps were performed in the presence of SOOmM

KCl. For the third sample the E. coli extract was incubated with the Ni-NTA agarose in the presence of 500mM potassium chloride, then proteins eluted in the presence

potassium glutamate. For the fourth sample the ionic conditions were similarly changed,

in this case, from SOOmM ammonium sulphate to potassium glutamate. For all samples,

and elution steps with SOOmM Imidazole (1300). The other buffer components are

described in materials and methods.

Figure 15a shows the results of SDS PAGE electrophoresis and analysis by

immunoblotting, for samples from each step of the purifications. In each case samples of

cell extract (charge) were taken before (1) and after (2) a centrifugation step, to assess

rCdc6p solubility. Samples were also taken from the flow through and washes of each

purification. The eluate contains proteins removed from the resin by incubation with

SOOmM Imidazole.

rCdc6p was not detected in the elution of Sample 1, where all steps of purification

were performed in the presence of potassium glutamate. This result was consistent with

Figure 14b, and reconfirms that rCdc6p does not bind Ni-NTA agarose in the presence of

600mM potassium glutamate. rCdc6 was detected in the flow through of the incubation,

which is consistent with this idea.

In sample 2, where potassium chloride was used for all steps o f the purification, a

strong signal for rCdc6p was detected in the flow through. However, Cdc6p was also

detected in the elute of this column, demonstrating, that the protein associated the Ni-

NTA agarose and was eluted with SOOmM Imidazole. The Cdc6p detected in the flow

through of the column may indicate that the concentration used was higher than the

capacity of Ni-NTA agarose.

Cdc6p was detected in the washes and elution of Sample 3, the highest

concentrations of Cdc6p being detected in the second wash and elution. In this sample,

the incubation o f the extract with resin and the first wash was performed in the presence

Figure 15 rCdc6p from BL21-Codon Plus™ pET15b-C7)C6, purified

using Ni-NTA in the presence of different ions.

A Ni-NTA chromatography.

rCdc6p was induced from BL21-Codon Plus^“ pET15b-C£)Cd as described previously. The Cell pellet was lysed, then split into 4 different samples. Ion concentrations were adjusted by the addition o f different buffers, as follows: ( i) KGI20 (5mM p- mercaptoethanol, 20mM imidazole, 40mM hepes-KOH pH 7.8, 160mM sorbitol, 0.1% triton xlOO, 2mM magnesium acetate, 0.25mM AEBSF, 600mM potassium glutamate), (2) KC1I20 (50mM Hepes pH7.8, 0.1% NP40, 10% glycerol, 20mM imidazoleSOOmM, potassium chloride), (3) KC1I20, (4) ASI20(5mM P-mercaptoethanol, 20mM imidazole, 40mM hepes-KOH pH 7.8, 160mM sorbitol, 0.1% triton xlOO, 2mM magnesium acetate, 0.25mM AEBSF, SOOmM ammonium sulphate).

A sample of each extract was taken before and after a centrifugation step to determine rCdc6p solubility (Charge 1/Charge 2). Each charge was then incubated with Ni-NTA agarose in small scale incubations that are described in section 2.6.5. The resin was washed twice and a step elution performed with 300mM imidazole. The buffers for elution were: (i) KGI300, (2) KC1I300, (3) KGI300, (4) KGI300. Samples from each step of the purifications were subjected to electrophoresis on a SDS page gel then immunoblotting with a monoclonal for Cdc6p.

B Activity for pre-RC assembly.

Loading Assays were performed with W and A' ARSl beads, Cdc6p free ammonium sulphate cut extracts and rCdc6p samples from different purification procedures (after dialysis of each againt KG600): (A) rCdcbp produced from the FB810 pETlSb-CDCd (FB810) with KGI20 buffer as described in Figure 14a. (2, 3, 4) rCdc6p produced from BL21-Codon Plus^“ pETlSb-CDCd as described above.

C The Supernatants

15A rCdc6p — ^ — 15 B 15C Strain FB810 Codon + Salt A 2 3 4 ARSl W A W A- W A W A rCdc6p Mcm2p » <* Supernatants Strain FB810 Codon + Salt A 2 3 4 ARSl W A W A- W A W A rCdc6p

elution. The results indicate that when the buffer was changed, a proportion of Cdc6p

was eluted (Wash 2), before the concentration of imidazole was increased. Despite this

loss Cdc6p was detected in the imidazole elution.

In Sample 4 Cdc6p was again detected in the second wash and elute. In this

sample the incubation and first wash were performed in the presence of ammonium

sulphate, then the second wash and elute in the presence o f potassium glutamate. The

results from Samples 3 and 4 suggest that changing the anions after the incubation of

extracts with Ni-NTA agarose, causes an elution of protein from Ni-NTA, independent of

imidazole concentration. In both cases however, some Cdc6p remained bound to the

resin and was eluted when the concentration o f imidazole was increased for the elution

step.

In conclusion, rCdc6p from BL21-Codon Plus'^’^ pET15b-CDCd does not bind

Ni-NTA agarose resin in the presence of SOOmM potassium glutamate. Binding does

occur in the presence o f SOOmM potassium chloride or SOOmM ammonium sulphate.

Changing the anion present or increasing the concentration of Imidazole to 300mM

induces elution of rCdc6p from the resin. In the next section we performed loading

assays with the different rCdc6p elutions obtained from this experiment, to investigate