Optimisation of expression and affinity purification strategies for the

4.2 Results

4.2.2 Optimisation of expression and affinity purification strategies for the

The rPfLDH and rPvLDH proteins were expressed from a pKK223-3 vector, with a His tag introduced during the cloning procedure, allowing for the TALON^® Co²⁺ purification, where rPyLDH was expressed from a pET-28a(+) plasmid. All growth curves had similar trends, where the TB cultures for all three proteins had the fastest growth rates to O.D. ~0.5 as shown in Figures 4.2 to 4.4 (A). The rPyLDH TB cultures had an initial O.D. that was higher than the LB cultures and maintained this difference in O.D. even during the stationary growth phase till around the tenth hour after inoculation. The rPfLDH cultures were the only cultures with a noticeable lag phase after inoculation.

For rPfLDH the highest 280 nm absorbance values were observed for the TALON^®(Co²⁺) resin elution profiles of the induced LB cultures grown at 37°C and both the 30 and 37°C TB cultures. With regard to rPvLDH the best elution profiles were attained for the TB cultures and the induced 37°C LB culture (Figure 4.3 (B)). The TB cultures expressing rPyLDH gave the highest absorbance readings in this case (Figure 4.2 to 4.4 (B)).

Analysis of the eluents on SDS-PAGE showed that some cultures had more co-purifying proteins, but these were also limited to the first two to three eluents in each case, which correlated to the elution of the E. coli host proteins seen in Figure 4.1 (C). It should be noted that the purifications performed here did not include imidazole in the wash steps. Some leaky expression was observed for all three proteins with cultures grown in LB media without IPTG induction.

Culture Growth conditions Eluent peak # Imidazole (mM)

BL21(DE3) LB 30°C 4 80

LB 30°C + IPTG 3 50

TB 30°C 5 100

LB 37°C 5 100

LB 37°C + IPTG 4 80

TB 37°C 5 100

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16

A 600nm

Time (hrs)

0 100 200 300 400 500

0 0.5 1 1.5 2 2.5 3

0 sn unbound wash 1 wash 2 wash 3 wash 4 eluent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 [Imidazole] (mM)

A 280nm

Eluent

rPfLDH LB 30 rPfLDH LB IPTG 30 rPfLDH TB 30 rPfLDH LB 37 rPfLDH LB IPTG 37 rPfLDH TB 37 Imidazole

11666.2 45 35 25 18.414.4 (kD)

30 °C LB 30 °C LB + IPTG 30 °C TB 37 °C LB 37 °C LB + IPTG 37 °C TB

1 3 5 7

Mw 2 4 6 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 56 7 8 9

30 °C LB 30 °C LB+IPTG 30 °C TB 37 °C LB 37 °C LB+IPTG 37 °C TB

IPTG addition

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16

A 600nm

Time (hrs)

0 100 200 300 400 500

0 0.5 1 1.5 2 2.5 3

0 sn

unbound wash 1 wash 2 wash 3 wash 4 eluent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

[Imidazole] (mM)

A 280nm

Eluent

rPfLDH LB 30 rPfLDH LB IPTG 30 rPfLDH TB 30 rPfLDH LB 37 rPfLDH LB IPTG 37 rPfLDH TB 37 Imidazole

A B

Figure 4.2 Growth of rPfLDH expressing cultures under varying conditions and elution profiles off a TALON^® (Co²⁺) resin analysed by SDS-PAGE

Growth curves of the expression host E. coli strain BL21(DE3) expressing rPfLDH (pKK223-3 vector) in different culture media at 30 and 37°C were plotted, with the addition of IPTG for induced cultures at O.D. ~ 0.5 indicated on the y-axis in (A). Each of the cultures were lysed by sonication and incubated on 1 ml packed TALON^® (Co²⁺) resin and the bound rPfLDH was eluted with a linear imidazole gradient from 0 to 500 mM as shown on the secondary axis in (B), eluting protein measured at 280 nm on the primary axis. The key to both (A) and (B) was shown between the graphs. Lysates and eluents of the corresponding cultures plotted in (A and B) were run on 12.5%

reducing SDS-PAGE gels and Coomassie stained (C). The samples run in each lane were as follows: molecular weight marker (Mw); original sample lysate loaded onto the TALON^® resin (lane 1); unbound sample (lane 2); final wash (lanes 3); eluents 1 to 6 (lanes 4 to 9).

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16

A 600nm

Time (hrs)

0 100 200 300 400 500

0 0.5 1 1.5 2 2.5 3

0 sn unbound wash 1 wash 2 wash 3 wash 4 eluent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 [Imidazole] (mM)

A 280nm

Eluent

rPvLDH LB 30 rPvLDH LB IPTG 30 rPvLDH TB 30 rPvLDH LB 37 rPvLDH LB IPTG 37 rPvLDH TB 37 Imidazole

116 66.2 45 35 25 18.4 14.4 (kD)

30 °C LB 30 °C LB + IPTG 30 °C TB 37 °C LB 37 °C LB + IPTG 37 °C TB

A B

1 3 5 7

Mw 2 4 6 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

30°C LB 30°C LB+IPTG 30°C TB 37°C LB 37°C LB+IPTG 37°C TB

IPTG addition

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16

A 600nm

Time (hrs)

0 100 200 300 400 500

0 0.5 1 1.5 2 2.5 3

0 sn

unbound wash 1 wash 2 wash 3 wash 4 eluent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

[Imidazole] (mM)

A 280nm

Eluent

rPvLDH LB 30 rPvLDH LB IPTG 30 rPvLDH TB 30 rPvLDH LB 37 rPvLDH LB IPTG 37 rPvLDH TB 37 Imidazole

A B

Figure 4.3 Growth of rPvLDH expressing cultures under varying conditions and elution profiles off a TALON^® (Co²⁺) resin analysed by SDS-PAGE

Growth curves of the expression host E. coli strain BL21(DE3) expressing rPvLDH (pKK223-3 vector) in different culture media at 30 and 37°C were plotted, with the addition of IPTG for induced cultures at O.D. ~ 0.5 indicated on the y-axis in (A). Each of the cultures were lysed by sonication and incubated on 1 ml packed TALON^® (Co²⁺) resin and the bound rPvLDH was eluted with a linear imidazole gradient from 0 to 500 mM as shown on the secondary axis in (B), eluting protein measured at 280 nm on the primary axis. The key to both (A) and (B) was shown between the graphs. Lysates and eluents of the corresponding cultures plotted in (A and B) were run on 12.5%

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16

A 600nm

Time (hrs)

0 100 200 300 400 500

0 0.5 1 1.5 2 2.5 3

0 sn unbound wash 1 wash 2 wash 3 wash 4 eluent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 [Imidazole] (mM)

A 280nm

Eluent

rPyLDH LB 30 rPyLDH LB IPTG 30 rPyLDH TB 30 rPyLDH LB 37 rPyLDH LB IPTG 37 rPyLDH TB 37 Imidazole

11666.2 45 35 25 18.414.4 (kD)

30 °C LB 30 °C LB + IPTG 30 °C TB 37 °C LB 37 °C LB + IPTG 37 °C TB

1 3 5 7

Mw 2 4 6 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9 Mw 1 2 3 4 5 6 7 8 9

30°C LB 30°C LB+IPTG 30°C TB 37°C LB 37°C LB+IPTG 37°C TB

IPTG addition

0 0.5 1 1.5 2 2.5

0 2 4 6 8 10 12 14 16

A 600nm

Time (hrs)

0 100 200 300 400 500

0 0.5 1 1.5 2 2.5 3

0 sn unbound wash 1 wash 2 wash 3 wash 4 eluent 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 [Imidazole] (mM)

A 280nm

Eluent

rPyLDH LB 30 rPyLDH LB IPTG 30 rPyLDH TB 30 rPyLDH LB 37 rPyLDH LB IPTG 37 rPyLDH TB 37 Imidazole

A B

Figure 4.4 Growth of rPyLDH expressing cultures under varying conditions and elution profiles off a TALON^® (Co²⁺) resin analysed by SDS-PAGE

Growth curves of the expression host E. coli strain BL21(DE3) expressing rPyLDH (pET-28a(+) vector) in different culture media at 30 and 37°C were plotted, with the addition of IPTG for induced cultures at O.D. ~ 0.5 indicated on the y-axis in (A). Each of the cultures were lysed by sonication and incubated on 1 ml packed TALON^® (Co²⁺) resin and the bound rPyLDH was eluted with a linear imidazole gradient from 0 to 500 mM as shown on the secondary axis in (B), eluting protein measured at 280 nm on the primary axis. The key to both (A) and (B) was shown between the graphs. Lysates and eluents of the corresponding cultures plotted in (A and B) were run on 12.5%

97 The imidazole concentrations required to elute the respective Plasmodium LDH proteins expressed here off the TALON^® (Co²⁺) resin are summarised in Table 4.3 below.

Table 4.3 Imidazole concentrations required for elution of bound recombinant Plasmodium LDH proteins off the TALON^® (Co²⁺) resin

Growth conditions rPfLDH

[Imidazole] (mM) rPvLDH

[Imidazole] (mM) rPyLDH [Imidazole] (mM)

LB 30°C 80 80 80

LB 30°C + IPTG 80 80 80

TB 30°C 50 80 80

LB 37°C 80 80 50

LB 37°C + IPTG 50 80 100

TB 37°C 80 80 80

Recombinant P. falciparum LDH eluted off the TALON^® (Co²⁺) resin at imidazole concentrations between 50 to 80 mM; rPvLDH at 80 mM and rPyLDH between 50 to 100 mM, dependant on the conditions used for expression.

4.2.3 Optimisation of expression and affinity purification strategies for the

Dalam dokumen Developing antibodies against Plasmodium lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and phosphoethanolamine-N-methyltransferase (Halaman 113-117)