Chapter 3: Molecular chaperone co-expression of Trypanosoma congolense M1
3.3 Results
3.3.2 Molecular chaperone co-expression
Five different molecular chaperones were introduced for each target protein and co-expressed to improve the recombinant production of soluble aminopeptidases. The 12.5% reducing SDS- PAGE analysis of the molecular chaperone expressing E. coli cells showed bands in the soluble fractions corresponding to the molecular chaperones at the expected sizes i. e. DnaK at ~70 kDa, GroEL at ~60 kDa, TF at ~56 kDa, DnaJ at ~40 kDa, and GrpE at ~22 kDa which were not in the control samples (NC) that contained TcoAP1 or TcoAP2 expressing cells alone in Figure 3.6 and 3.7, respectively.
Figure 3.6: TcoAP1 and chaperone co-expression 12.5% reducing SDS-PAGE analysis. TcoAP1 was co-expressed with several molecular chaperones then samples from the cell lysate (L) and soluble fraction (S) were analysed by 12.5% reducing SDS-PAGE stained with Coomassie blue R-250. M: Pre- stained protein marker, NC: No chaperones.
Co-expression of TcoAP1 with the molecular chaperones was analysed using 12.5% reducing SDS-PAGE, as shown in Figure 3.6, even with the presence of significant amounts of overexpressed molecular chaperones, most of TcoAP1 was found within the insoluble fraction.
A similar co-expression profile was seen for TcoAP2, were most of the protein was still found in the insoluble fraction (Figure 3.7).
M
NC pKJE7 pKJE8 pGro7 pG-Tf2 pTf16
97 68 45
30
21
L S L S L S L S L S L S
TcoAP1, 91 kDa DnaK
GroEL TF
DnaJ GrpE kDa
69 Figure 3.7: TcoAP2 and chaperone co-expression 12.5% reducing SDS-PAGE analysis. TcoAP1 was co-expressed with several molecular chaperones then samples from the cell lysate (L) and soluble fraction (S) were analysed by 12.5% reducing SDS-PAGE stained with Coomassie blue R-250. M: Pre- stained protein marker, NC: No chaperones.
In Figure 3.8, A, another 12.5% reducing SDS-PAGE analysis of the co-expression experiments was performed, this time only soluble fractions of the co-expression samples were loaded as well as the cell lysate of TcoAP1 recombinant expression without chaperones.
Furthermore, western blot analysis was performed to detect only the His6-tagged recombinant protein (Figure 3.8, B), for a more sensitive and specific analysis. In Figure 3.8, A, the production of soluble TcoAP1 in conjunction with the molecular chaperones was compared with the whole cell lysate TcoAP1 sample which was not co-expressed with the chaperones.
The production of soluble TcoAP1 seems almost negligible when compared to the total TcoAP1 found in the cell lysate when viewing the 12.5% reducing SDS- PAGE gel. However, western blot analysis of the soluble TcoAP1 co-expression samples (Figure 3.8, B) showed a visible increase in soluble TcoAP1 protein expression compared to the soluble fraction of the TcoAP1 expression without molecular chaperones (NC). Furthermore, co-expression of TcoAP1 with the pKJE7 chaperones (DnaK-DnaJ-GrpE) led to the biggest improvement in soluble TcoAP1 expression (Figure 3.9).
M
pKJE7 pKJE8 pGro7 pG-Tf2 pTf16 NC
L S L S L S L S L S L S
97 68 45
30
GroEL TF DnaJ DnaK
TcoAP2, 91 kDa
GrpE kDa
70 Figure 3.8: 12.5% reducing SDS-PAGE and western blot analysis of soluble recombinant TcoAP1 after molecular chaperone co-expression. Samples of the soluble fractions and a reference cell lysate sample of recombinant TcoAP1 co-expression with five different molecular chaperone teams were electrophoresed on 12.5% reducing SDS-PAGE gels and (A) stained with Coomassie blue R-250 while (B) was transferred onto nitrocellulose and incubated with chicken anti-His6 HRPO conjugate [1:4000 in 0.5% (w/v) BSA-PBS]. M: Pre-stained protein marker. NC: No chaperones.
Figure 3.9: Comparison of recombinant TcoAP1 solubility after molecular chaperone co- expression. The amount of soluble recombinant TcoAP1 were compared according to band intensities of the western blot using GelAnalyzer®. NC: No chaperones.
In Figure 3.10, Panel A, another 12.5% reducing SDS-PAGE analysis of the co-expression performed is shown, again with only the soluble fractions to analyse soluble recombinant
A
B 120
85
50
35
25
DnaK GroEL TF
DnaJ
TcoAP1, 91 kDa
GrpE
M NC pKJE8 pKJE7
pGro7
pG-Tf2 pTf16
Cell lysate
kDa
TcoAP1, 91 kDa
0 5 10 15 20 25 30
NC pG-KJE8 pKJE7 pTf16 pG-Tf2 pGro7
Soluble proportion of rTcoAP1 (%)
Molecular chaperone co-expression teams
71 TcoAP2 expression, western blot analysis was also performed to detect only the His6-tagged recombinant protein (Figure 3.10, B). The production of soluble TcoAP2 in conjunction with the molecular chaperones was compared with the whole cell lysate TcoAP2 sample which was not co-expressed with the chaperones. The production of soluble TcoAP2 also seemed negligible when compared to the total TcoAP2 found in the cell lysate when viewing the 12.5%
reducing SDS-PAGE gel (Figure 3.10, A). However, western blot analysis of the soluble TcoAP2 co-expression samples (Figure 3.10, B) showed, as in the case of TcoAP1, a visible increase in soluble TcoAP2 protein expression compared to the soluble fraction of the TcoAP2 expression without molecular chaperones (NC). Again, co-expression of TcoAP2 with the pKJE7 chaperones (DnaK-DnaJ-GrpE) led to the biggest improvement in soluble TcoAP2 expression (Figure 3.11).
Figure 3.10: 12.5% reducing SDS-PAGE and western blot analysis of soluble recombinant TcoAP2 after molecular chaperone co-expression. Samples of the soluble fractions and a reference cell lysate sample of recombinant TcoAP1 co-expression with five different molecular chaperone teams were electrophoresed on 12.5% reducing SDS-PAGE gels and (A) stained with Coomassie blue R-250 while (B) was transferred onto nitrocellulose and incubated with chicken anti-His6 HRPO conjugate [1:4000 in 0.5% (w/v) BSA-PBS]. M: Pre-stained protein marker. NC: No chaperones.
A
B
M NC pKJE8 pKJE7
pGro7
pG-Tf2 pTf16
Cell lysate
DnaK GroEL TF
DnaJ
TcoAP2, 91 kDa
GrpE 120
85
50
35
25 kDa
TcoAP2, 91 kDa
72 Figure 3.11: Comparison of recombinant TcoAP2 solubility after molecular chaperone co-
expression. The amount of soluble recombinant TcoAP2 were compared according to band intensities of the western blot using GelAnalyzer®. NC: No chaperones.
3.3.3 Solubilisation, refolding and purification of recombinant TcoAP1 and TcoAP2