CHAPTER 3 DETECTION OF PLASMODIUM YOELII (17XL) ACETYL-COA
3.3 Discussion
5.2.9 Large scale expression, purification and
In order to obtain a sizable amount of the recombinant His6-PyBCCP to be used for oligomerisation studies, expression of His6-PyBCCP was carried out on a larger scale. A culture (4.5 L) of E. coli cells expressing His6-PyBCCP was induced with 0.5 mM IPTG for 5 h at 37°C. His6-PyBCCP proteins in the inclusion bodies were separated with an immobilised-cobalt affinity matrix under denaturing conditions. Figure 5.17A illustrates the SDS-PAGE analysis of the purification profile of His6-PyBCCP under denaturing conditions.
Lane 1 is the insoluble fraction that was subsequently purified over the immobilised-cobalt affinity matrix and lane 2 contains the supernatant of the 2 M urea wash step prior to guanidine solubilisation (see Section 2.6.2). It can be seen that the prominent band at 50 kD corresponding to the expressed protein was predominantly in the insoluble fraction and was not solubilised by the 2 M urea wash solution. Lanes 3 – 5 contain no visible protein bands from the wash steps after binding to the immobilised-cobalt affinity matrix (prior to elution).
Figure 5.14 Analysis of the in vivo biotinylation of His6-PyBCCP. Total cell lysate from E. coli cells expressing His6-PyBCCP induced with IPTG (+) or uninduced (−), in the presence (+) or absence (−) of biotin, was resolved by SDS-PAGE (10%), blotted on a nitrocellulose membrane and probed with avidin-peroxidase alone (A) or incubated with free avidin followed by avidin-peroxidase conjugate (B) or incubated with avidin-peroxidase conjugate in the presence of free biotin (C).
Mw 1 2 3 4
IPTG –
+
–+
Biotin –
–
+ +
Mw 1 2 3 4
–
+
–+
–
–
+ +
Mw 1 2 3 4
–
+
–+
–
–
+ +
A B C
kD 116.0
66.2
45.0
35.0
25.0
18.4
128 Figure 5.15 Analysis of the in vivo biotinylation of GST-PyBCCP. Total cell proteins from E. coli cells expressing GST-PyBCCP induced with IPTG (+) or uninduced (−), in the presence (+) or absence (−) of biotin, was resolved by SDS-PAGE (10%), blotted on a nitrocellulose membrane and probed with avidin- peroxidase conjugate alone (A) or incubated with free avidin followed by avidin-peroxidase conjugate (B) or incubated with avidin-peroxidase conjugate in the presence of free biotin (C).
Mw 1 2 3 4
IPTG –
+
–+
Biotin –
–
+ +
Mw 1 2 3 4
–
+
–+
–
–
+ +
Mw 1 2 3 4
–
+
–+
–
–
+ +
A B C
kD
116.0
66.2
45.0
35.0
25.0
18.4
Lane 6 contains proteins eluted with 1 M imidazole in 8 M urea buffer. The dominant protein band in lane 6 matches the 50 kD band in lane 1 corresponding to the recombinant His6- PyBCCP. Proteins that did not bind to the immobilised-cobalt affinity matrix (before the wash steps) were not resolved on the acrylamide gel because guanidine forms an insoluble complex with SDS in the treatment buffer. Figure 5.17B illustrates the SDS-PAGE analysis of the purification profile of His6-PyBCCP under native conditions using the soluble fraction obtained from cell culture (500 ml) induced with 0.5 mM IPTG (16 h, 18°C). This condition was not used because the level of expression was more than 100-fold less compared to expression at 37°C, though the expression at 18°C resulted in recombinant proteins being in the soluble fraction. Figure 5.17B also demonstrated that His6-PyBCCP expressed at 18°C was capable of binding to the immobilised-cobalt affinity matrix.
The purified His6-PyBCCP was renatured by a series of dialysis steps against decreasing concentrations of urea in 20 mM Tris-HCl buffer. At 0.5 M urea, part of the protein precipitated out of the solution. Thus, some of the recombinant protein was lost during the refolding process. The insoluble protein and the refolded protein were resolved along side Figure 5.16 Analysis of the in vivo biotinylation of GST-PyCTRNt. Total cell lysate from E.
coli cells expressing GST-PyCTRNt induced with IPTG (+) or uninduced (−), in the presence (+) or absence (−) of biotin, was resolved by SDS-PAGE (10%), blotted on a nitrocellulose membrane and probed with avidin-peroxidase conjugate.
Mw 1 2 3 4
IPTG –
+
–+
Biotin –
–
+ +
kD
116.0
66.2
45.0
35.0
25.0
18.4
each other on 10% SDS-PAGE (Figure 5.18). The results showed that part of the refolded protein formed precipitates; however, a substantial amount of the recombinant protein was renatured and solubilised. Approximately 10 mg of recombinant purified recombinant protein per litre of culture was obtained.
Figure 5.17 Immobilised-cobalt affinity purification of His6-PyBCCP. The purification profile of recombinant His6-PyBCCP was analysed by SDS-PAGE. (A) Under denaturing conditions (10%
acrylamide gel). Lane 1, insoluble fraction; lanes 2 – 5, wash steps; lane 6, elution with 1 M imidazole. (B) Under native condition (12.5% acrylamide gel). Lane 1, soluble fraction of the cell lysate; lane 2, flow through fraction; lanes 3 and 4 first and second wash steps, respectively; lane 5 and 6, elution with 50 mM and 250 mM imidazole respectively.
Mw 1 2 3 4 5 6 kD
116.0
66.2
45.0
35.0
25.0
18.4 A
Mw 1 2 3 4 5 6 kD
116.0
66.2
45.0
35.0
25.0
18.4
14.4 B
The refolded protein was analysed by non-reducing SDS-PAGE (10%) to ascertain if the recombinant protein formed oligomer in the absence of β-mercaptoethanol or DTT (Figure 5.19). The result showed that the recombinant protein did not form a defined quaternary structure but rather aggregated and did not resolve in the running gel (lane 2) when compared to the reduced recombinant protein sample (lane 1).
kD
116.0
66.2
45.0
35.0
25.0
18.4
Mw 1 2
Figure 5.19 SDS-PAGE analysis of the oligomerisation of His6-PyBCCP. Purified and refolded His6-PyBCCP was treated with reduced with β-mercpatoethanol (lane 1) or not reduced (lane 2) and resolved by SDS-PAGE (10%).
kD
116.0
66.2
45.0
35.0
25.0
18.4
Mw 1 2 3
Figure 5.18 Refolding of immobilised-cobalt affinity purified-His6-PyBCCP. The refolding of the purified recombinant His6-PyBCCP was resolved by SDS-PAGE (10%). Lane 1, starting material; lane 2, precipitated product; lane 3, solubilised recombinant protein.
A similar result was obtained when the acrylamide concentration was reduced to 5.5%, low enough to resolve proteins between 45 kD and 350 kD (result not shown). There is no data that suggests the precise size of the polymeric form of acetyl-CoA carboxylase. Probably, the aghregation of the recombinant protein may be due to the presence of cystine residues, which is reduced in the presence of β-mercaptoethanol.