Predicción de sitios de unión a TyrR en el genoma de A brasilense Sp7

lEF (see Figure 3.3 A for explanation o f event sequence). Whether this was attempted stepwise with 6M protein into 5M, 4M, 3M etc. levels o f urea or rapidly, into OM

urea, the end result was identical; complete precipitation o f the protein with none left in solution (as evidenced by protein estimation and also as visualised by SDS-PAGE). The precipitated material could not be resolubilised into urea.

After the lEF step, investigations into resolubilisation branched out. As had been attempted with the non-IEF treated MRP-8, either a stepwise or rapid protocol

was followed. Both protocols resulted in a significant amount o f protein precipitation but significantly, there appeared to be soluble material remaining (by protein

estimation). Therefore, the final step in the human protocol, purification on hydroxyapatite was attempted.

Hydrox>^apatite binds only calcium binding proteins. Therefore, it was ideal for the purification o f EF-hand containing proteins such as M RP- 8 and M R P-14. This

selection step had a second level o f complexity in that it would clearly only bind functional calcium binding proteins, that is, correctly folded proteins. Therefore, a protein that had not folded correctly would not bind to the column and would flow through to waste. This assumption produced an observation about the refolding

Chapter 3:- Cloning and purification o f recombinant M RP- 8 and M RP-14

protocols that had been attempted; the rapid renaturation protocol produced an approximately double flow through o f material compared to the slow renaturation protocol, for a given amount o f starting material (data not shown). This suggested that the rapid renaturation protocol produced a higher percentage o f incorrectly folded material than did the slow renaturation protocol. The end result o f the hydroxyapatite purification o f M RP- 8 from either renaturation protocol was a peak visible to the

spectrophotometer at 25-30%B and 280nm. However, this material was not visible on SDS-PAGE gels stained with coomassie or silver stain. Protein estimation suggested the presence o f very small amounts o f material (200|Xg/0.5L with slow renaturation and 80|uig/0.5L with fast renaturation). Whether this was real or an artefact o f the protein estimation system, given that the levels were so low, remains to be seen. It is thought that the difference between the initial spectrophotometer readings and the later gel analysis results were due to precipitation rather than a buffer effect as HA-B buffer does not absorb at 280nm. Whatever the answer was, this method was not going to produce material stable enough to work with, or enough material for functional assays or immunisations.

Given that some soluble MRP- 8 was produced in the E.coli and that the

hydroxyapetite column system was able to select for calcium-binding proteins, an attempt was made to purify soluble M RP-8, directly from the bacterial cytosol, thereby

obviating any resolubilisation problems. The original hypothesis was that only MRP- 8

would be bound to the column. Therefore, in theory a large amount o f bacterial product could be added to the column to make up for the low cytosolic expression. A cytosolic preparation was made from an induced bacterial pellet and this was initially purified by centrifugation and 0.45|xm filtration. After loading onto a hydroxyapetite column was performed, soluble M RP- 8 was retrieved, eluting at 25%B (125mM phosphate), but it

was approximately 10% pure, (see Figure 3.3D, lanes marked compare with ‘ 8 SN’

in Figure 3.3B). Moreover, the protocol was clumsy as there was a very long wash period to remove non-specifically associated bacterial proteins from the column. Therefore, it was decided that this protocol was also not viable for the production o f M R P -8 .

No mention has been made in this description o f the attempted purification o f M RP-8, about the M RP- 8 mutant, M RP-8FY. Purification attempts were not made for

Chapter 3:- Cloning and purification o f recombinant MRP-8 and M RP-14 The one hopeful aspect about the purification o f M RP- 8 was the seeming fluid

phase stability o f the molecule, that is to say, the ability to relatively purify the molecule from the bacterial cytosol without precipitation. This observation produced that idea that the way forward would be to obviate the negative features o f

hydroxyapatite purification o f bacterial cytosol i.e. the contamination. The best way to do this was to attempt purification o f MRP-8, after affinity tagging.

3.3.2 pET-28a

After the relative failure o f either MRP- 8 or M RP-14 (see section 3.4.1) purification

using an untagged recombinant protein, the decision was taken to tag the molecules for purification. This was not initially attempted as proteins cloned in tagged vectors are either permanently fused to the tags or, after tag removal, a number o f additional amino acids remain that could potentially alter protein function. However, molecule tagging seemed to be the only way forward and so the molecules were conveniently cloned into pET-28a, a His tagged vector. This vector allows either N- or C- terminal his-tagging with 6 consecutive His residues. Both molecules were N-terminally tagged because (a) it

was easier as the sites were exactly the same in pET-3a and (b) this allowed tag removal by thrombin, although this leaves the following 3 amino acids, before the methionine o f the protein o f interest, Gly-Ser-His. C-terminal tagging was not considered as it had various disadvantages, including an inability to remove the His-tag after protein production.

The initial step in the purification o f His-MRP- 8 was to evaluate the subcellular

localisation; this was found to be split evenly between the inclusion bodies and the cytosol (see Figure 3.4B, on page 117). The optimal conditions for cytosolic expression o f M RP- 8 appeared to be 0.4mM IPTG for 5 hours at 37°C; optimal M RP-8FY

expression was identical except that the optimal incubation temperature was 28°C. Increasing IPTG concentrations up to ImM did not increase the amount o f induction, as visualised by SDS-PAGE (data not shown).

Tagging MRP- 8 had a profound effect on the calculated lEP, moving it from 5.55

to 6 .8, perhaps this change is associated with the relative increase in protein solubility

(compare to untagged M RP- 8 in Figure 3.3B). Interestingly, M RP- 8 was still

Chapter 3:- Cloning and purification o f recombinant M RP- 8 and M RP-14

SDS-PAGE (MRP- 8 from lOkDa to 19kDa, and M RP-8FY from 6kDa to 10.5kDa)

(see Figure 3.4B, on page 117).

Purification o f His-MRP- 8 from bacterial supernatant was relatively

straightforward. An overview o f the steps taken is described in Figure 3.4 A. Although the protein could be purified to homogeneity (Figure 3.4C) eluting at 27-30%B, the yield o f protein, at 0.5mg/L (starting culture) was low. Furthermore, this protein precipitated within 6 days, when stored at 4°C and overnight if left at room

temperature.

Attempts to cleave the tag from the protein resulted in complete precipitation o f the protein, if the cleavage was performed at 37°C and approximately 50% if performed at room temperature. As the yield prior to this step was 0.5mg/L, progression towards cleaved, purified protein, dialysed into an assay buffer that would not affect cells was increasingly difficult. Attempts to dialyse the His-tagged protein from the final imidazole and salt rich buffer were also relatively unsuccessful; as evident from precipitation and a recovery o f approximately 1 0 0|ig from a Im g pre-dialysis starting

point, followed by further protein precipitation over 48h. A t this point, the attempts to generate murine M RP- 8 in order to confirm the results o f the Geczy group (137)

were abandoned. Protein was henceforth routinely generated as a His-tagged product in order to immunise rabbits, every time an immunogenic boost was required.

A