Cloning of mature yeast mitochondrial aconitase and co-expression with GroEL/ES chaperones
A 2.4 kb mature ACO1 gene was amplified from the template plasmid pQE60Aco by polymerase chain reaction and cloned downstream of the T7lac promoter in pET29 vector between the restriction sites NdeI and XhoI. The resulting plasmid was designated as pETAco. The plasmid pETAco was double digested with restriction enzymes NdeI and XhoI to get the “pop-out” of the gene of insert corresponding to size of 2.4 kb (Fig 1A). Further, the insertion of the mature aconitase gene was confirmed by automated gene sequencing of the plasmid with the T7 promoter and T7 terminator sequences as forward and reverse primers. Both results confirmed the cloning of the mature aconitase gene sequence in the pET29 vector. The expression of the aconitase gene was checked on SDS-PAGE gel of the BL21(DE3) cells transformed with the plasmid grown in LB media until mid-log phase and induced with 0.5mM IPTG (Fig 1B). The cells expressing aconitase were re-transformed with pGro for expression of GroEL/ES. The co-expression of both the yeast mitochondrial aconitase and GroEL/ES was verified on a 12% SDS-PAGE gel of total cell lysate from transformed BL21(DE3) cells induced with 0.5mM of sterile IPTG and 0.5g/L of L-arabinose (Fig 1C).
Effect of temperatures during chaperone-assisted folding in E. coli cells
Micro-organisms require an optimal temperature for growth below which the growth rate tends to decrease. When the BL21(DE3) cells producing recombinant aconitase were grown at 37°C, 30°C, 25°C and 16°C, a steady decline in the specific growth rate was observed with decrease in temperatures demonstrating longer lag phase (Fig 2A). The biomass concentration of the cells expressing only aconitase at all temperatures above 16°C were not compromised, however, at 16°C the biomass was decreased by 25 percent and growth rate decreased by 35%. The high rate of recombinant protein synthesis imposes a systemic stress on the cells which directly impacts their growth rates. In case of cells co-expressing recombinant chaperones GroEL/ES and aconitase, the biomass concentrations were found to be 10% higher than cells expressing only aconitase at all temperatures (Fig 2B). The specific growth rate within 3 hours after induction of the cells co-expressing GroEL/ES chaperone was slightly higher than the cells expressing only aconitase indicating that the chaperones GroEL/ES were able to modulate this systemic stress (Additional materials 1).
Estimation of aconitase expression levels in cells grown at various temperatures revealed that aconitase expression was highest at 37oC which decreased with decrease in temperature. GroEL/ES co-expression had no major effect on aconitase expression, which remained nearly the same as in cells expressing aconitase in the absence of GroEL/ES co-expression (Table 1). At lower incubation temperatures, however, the GroEL/ES expression increased which can be explained by the slower rate of arabinose utilisation by the cells at lower temperatures.
Table 1. Effect of temperature on aconitase and GroEL/ES co-expression
Temperature
(°C)
|
Aconitase expression (mg/ g DCW) in cells expressing
|
GroEL expression (mg/ g DCW) in
E. coli cells
|
Fold increase in GroEL/ES expression as compared to 37°C
|
Aconitase
|
Aconitase & GroEL/ES
|
37 °C
|
24.03
|
23.78
|
14.67
|
-
|
30 °C
|
23.68
|
22.90
|
24.40
|
1.66
|
25 °C
|
20.93
|
20.13
|
59.49
|
4.06
|
16 °C
|
18.42
|
18.07
|
84.19
|
5.74
|
The total expressed protein does not always reflect the amount of functionally active protein which is dependent on the ability of the protein to fold correctly. The functionally active protein can be estimated as the amount of protein which is in soluble form separated from the mis-folded non-functional protein aggregates, recovered as insoluble pellet fraction of the cell lysate. The SDS-PAGE gels images show the solubility analysis of the samples of the cell lysate as a whole and fractionated as soluble and insoluble components (Fig 3 A, B, C, D). The solubility of the recombinant aconitase was calculated as the fraction of the expressed protein present in the soluble cell lysate to the protein present in the total cell lysate. The aconitase yield increased rapidly reaching a peak within 2 hours of induction at 37oC. At 25oC, the yield increased slowly and spread over a period of 10 hours of induction proving the nascent polypeptide time to fold in the complex cellular environment. When aconitase expressing cells were grown at 16°C, the solubility of the recombinant proteins was improved by 14-fold as compared to cells grown at 37°C showing that the protein solubility tends to improve at lower temperatures. The solubility is further improved with GroEL/ES co-expression which helps in aconitase folding. The spontaneous folding of aconitase protein is severely hampered at 37°C amounting to merely 5% which improves to just 9% in the presence of GroEL/ES co-expression. The most significant improvement in aconitase solubility was observed at 25°C. The GroEL/ES assistance improved solubility at all temperatures except at 16°C. Highest chaperone assisted folding was achieved from 9% to 27% at 30°C (Figure 3 E).
Since only properly folded proteins are biologically functional, enzyme activity is the main determinant of a protein’s natural folding. Aconitase activity was improved by lowering the temperature but not to the same extent as solubility. At all temperatures the aconitase activity was enhanced in presence of GroEL/ES. The maximum change in folding and activity due to chaperone co-expression was recorded at 30°C, with improvements in solubility and activity of 190% and 60%, respectively (Table 2). Interestingly, the chaperone GroEL/ES expression level improved by 1.66-fold by lowering the temperature to 30°C as compared to 37°C without compromising the aconitase expression. The in-vivo solubility of aconitase in the cells grown at 25°C is found to be ~ 65% of the total aconitase expressed which is 11-fold higher compared to aconitase expressed in the cells grown at 37°C. Similarly, the recombinant aconitase activity in the E. coli cells grown at 25°C enhanced by 7-fold as compared to the activity in the cells grown at 37°C. At 25°C, the increment in the aconitase activity and solubility due to GroEL/ES co-expression is reported to be 21% and 18%, respectively (Fig 3F).
Table 2. Enhancement in solubility and activity during chaperone assisted folding in recombinant E. coli grown at different temperatures
Temperature
|
Percentage enhancement during GroEL/ES assisted folding compared to control cells (%)
|
Aconitase Solubility
|
Aconitase activity
|
37 °C
|
68.7
|
42.5
|
30 °C
|
189.1
|
58.3
|
25 °C
|
18.3
|
21.4
|
16 °C
|
0.9
|
5.1
|
Microscopic Imaging of morphologies of E coli cells
The over-expressed recombinant protein tends to form inclusion bodies which reach to the micron size range. In order to study whether inclusion bodies have any effect on the cell morphology, the induced cells grown at 37°C and 25°C were studied by phase-contrast and atomic force microscopy and compared against the control (uninduced) cells. Some of the cells grown at 37°C possessed more than one area of increased density denoting formation of multiple inclusion bodies. They are mostly localized towards the poles which has been reported to be driven by the macromolecular crowding in the cytosol [11]. The presence of recombinant aconitase sequestered as inclusion bodies is clearly visible in E. coli cells grown at 37°C both in the presence and absence of exogenous chaperones GroEL/ES (Fig 4 A and B). The black arrows indicated in the shows the presence of mass of inclusion body of recombinant aconitase in the cells grown at 37°C as a protrusion after 18 hours of induction compared to the uninduced control. No such significant change was observed in the morphology of the cells expressing aconitase at lower temperature (Fig 4 C and D). Atomic force microscopy photos clearly highlight the contrast between cells grown at 37°C and cells grown at 25°C, where cells grown at 37°C show a mass of inclusion bodies that is not seen in uninduced cells or cells grown at 25°C (Fig 4 E-G).
Effect of IPTG concentration on the aconitase expression and solubility
Aconitase expression induced from three different IPTG concentrations (0.5, 1 and 2mM) were analysed in presence and absence of molecular chaperone co-expression at 37°C and 25°C (Fig 5 A and B). Growth rate tends to decrease with increase in IPTG concentration. The growth kinetics improves on GroEL/ES co-expression when the molecular chaperone rescues the expressed proteins from misfolding.
At 37°C the exponential phase reaches fast and is short while at 25°C the exponential phase is longer giving protein time to fold. This is also reflected in the solubility profile which improved at lower temperature. The aconitase expressed by cells grown at 25°C showed better aconitase solubility as compared to those grown at 37°C. It was found that the residual arabinose concentration got completely depleted within 3 hours of induction with 0.5 g/L of arabinose when the cells were grown at 37 °C (data not shown). Therefore, enough GroEL/ES chaperones are not produced to fold the misfolded proteins leaving most of the protein in inclusion bodies at high temperatures. Under all conditions the aconitase solubility improved upon co-expression of GroEL/ES chaperone. While it improved very slightly at 37°C, 15% enhancement in the aconitase solubility irrespective of the inducer concentrations was obtained at 25°C.
Figure 6A and B depicts the aconitase yield obtained under different temperatures and varying inducer concentrations. A rapid increase in expression levels is observed in cells induced at 37°C leading to large protein accumulation within 2 hours of induction. Cells grown at 25°C on the other hand exhibit a slower rate of production but the accumulation extended over a period of 10 hours in the induction regime providing the nascent polypeptides enough time to fold in the complex cellular environment. It was seen that cells induced with high IPTG concentration (2 mM), showed a very high rate of synthesis during initial phase of induction at both the temperatures which retarded after 4 hours.
It is well established that cells exhibiting high rates of protein synthesis incur high intermolecular interactions arising out of macromolecular crowding within the cells leading to formation of protein aggregates. Lower temperatures allow slow expression rates for longer durations.Also, the value of knetic constants for intermolecular interactions are lower leading to lessar intermolecular interactions. The protein folding is further facilitated by molecular chaperones. As seen by the increment in aconitase activity by ~15% in cells grown at 25°C at all inducer concentrations.
The solubility of the recombinant protein was not affected by inducer concentration irrespective of temperature or Gro EL/ES expression. It was observed that only low temperature was the important factor and the cells exhibited improved solubility which was further improved in presence of GroEL/ES (Table 3). The aconitase activity reduced with increase in inducer concentrations. The cells treated with higher concentrations of IPTG at both temperatures showed detrimental effect on the aconitase activity. This confirms that at higher expression rates more a of the expressed protein lost functionality due to misfolding of the polypeptide (Fig 6 C and D).
Table 3. Effect of IPTG induction on aconitase solubility in the recombinant BL21(DE3) cells during chaperone-mediated folding
IPTG
Concentration (mM)
|
Temperature (°C)
|
Aconitase solubility in E. coli cells expressing (%)
|
Aconitase
|
Aconitase & GroEL/ES
|
0.5 mM
|
37 °C
|
4.7
|
8.9
|
|
25 °C
|
64.1
|
76.4
|
1 mM
|
37 °C
|
5.1
|
8.5
|
|
25 °C
|
66.7
|
75.7
|
2 mM
|
37 °C
|
5.0
|
5.4
|
|
25 °C
|
65.7
|
-
|
|
|
|
|
|
Titration of chaperone expression required for folding of aconitase
The co-expression of chaperones enables folding of aconitase but the expression of additional gene from an additional plasmid exerts further imbalance in the cellular metabolism and the proteins expressed compete for the tRNA pool of the cellular systems. This part of the experiments was carried out to demonstrate the effect of chaperone co-expression on the expression of the recombinant protein. The co-transformed E. coli cells were grown at 25°C till OD600 of 0.6 to 0.8 and simultaneously induced with IPTG (0.5mM) and varying arabinose concentrations ranging from 0.01 to 0.75 mg/ml for GroEL/ES expression to study its effect on aconitase expression, solubility and activity.
The GroEL/ES expression increased with increasing concentrations of arabinose reaching upto 75 mg/g DCW of GroEL/ES within 14 hours of induction (Table 4). The aconitase expression was decreased by a maximum of 10% when both proteins were expressed at the same time. Compared to control cells with no exogenous GroEL/ES expression, the cells induced with increasing arabinose concentrations showed both increased aconitase activity and solubility. An 82% increase in aconitase activity reaching upto 145 IU/mg DCW was achieved when the recombinant proteins were expressed at 25°C with 0.5% arabinose induction yielding ~60 mg/g DCW of exogenous GroEL/ES. The large improvement in aconitase activity at high GroEL/ES accumulation suggests that a lack of chaperones in the cellular environment prevents misfolded proteins from achieving their native structure (Fig 7).
Table 4. Effect of arabinose titration on expression of aconitase and GroEL/ES in BL21(DE3) cells
Arabinose Concentration % (w/v)
|
Aconitase yield
(mg/g DCW)
|
GroEL/ES yield
(mg/g DCW)
|
Control
|
10.73
|
-
|
0.01
|
8.8
|
17.1
|
0.05
|
9.11
|
25.6
|
0.1
|
8.9
|
28.4
|
0.5
|
9.33
|
58.3
|
0.75
|
8.98
|
-
|
Effect of induction at different growth phases on the recombinant protein expression and activity
The time of induction during the fermentation plays a critical role in the final yield and the quality of the recombinant proteins in E coli. The transformed E coli cells were induced to express aconitase and GroEL/ES simultaneously at different cell densities. After 12 hours of induction, the normalised quantity of cells was used to study the effect of induction at different growth rates. The induction of the culture at the early stage of growth, when the cells are growing rapidly, results in a low final biomass yield. However, if the culture was induced at later stages of growth, when the specific growth rate of the cells is lower, the specific product yield is reduced. It has been reported that the solubility of the recombinant product increased when the culture growing at lower temperature of incubation was induced to express at late-log growth phase [12].
When the culture was induced at low cell density, the induced cells grow at a fast growth rate and when induced at high cell density, the induced cells grow at a slow growth rate. The cells induced at slow growth rates show reduced recombinant aconitase expression and activity. The specific aconitase yield reduced by 40%, when the culture was induced at the biomass density of 1.7. The reduction in the product yield at low specific growth rates may be due to the nutrient exhaustion and non-availability of precursor molecules for the polypeptide synthesis. Our results indicate that there is no appreciable change in the aconitase expression until the cell density of the culture goes beyond 1.3 (Table 5).
The aconitase activity reduction in the cells growing at slow growth rate indicates that the expressed proteins failed to attain native structural conformation. The recombinant aconitase solubility was not compromised when induced at higher cell density (Fig 8). This indicates that the expressed proteins accumulate as soluble aggregates devoid of functionality. The optimal cell density for the induction of the recombinant aconitase and GroEL/ES to obtain soluble and functional recombinant proteins in BL21 (DE3) is reported to be 0.6.
Table 5. Aconitase expression yield in the cells induced at various phases of growth
Cell density at the time of induction (OD600nm)
|
Aconitase Expressed (mg/g DCW)
|
0.6
|
20.83
|
0.8
|
20.40
|
1.1
|
19.91
|
1.3
|
17.67
|
1.7
|
15.45
|
Effect of media components on recombinant aconitase activity and solubility during chaperone assisted folding
The transformed E. coli cells were grown at 25°C in various media to see how media components affected the recombinant protein's expression, solubility, and functionality during GroEL/ES chaperone co-expression. The results in figure 9 shows that the cells grown in media composed of richer nutrients displayed higher biomass yield and growth rates compared to cells grown in chemically defined media. The aconitase expression was significantly higher in enriched media as compared to defined media which showed very less improvement on chaperone co-expression (Table 6). The cells grown in media devoid of complex nutrient components displayed hindered aconitase solubility and activity. The aconitase solubility was very much affected in the cells grown in defined media where only about one-fourth of the expressed proteins were present in soluble form which was enhanced to 50% of the total expressed proteins upon chaperone co-expression. The aconitase activity is significantly lower as compared to cells grown in Luria Broth which could be due to the non-availability of the prosthetic group Fe4S4 for the aconitase activity in the cells grown in the defined media.
In cells grown in enriched medium, the specific aconitase activity was higher and about 60% of the expressed aconitase was in soluble form which increased to about 75-80% on chaperone co-expression. In minimal media, however, GroEL/ES assisted folding increased aconitase solubility by twofold. These set of experiments clearly demonstrate that media components are very important for the quality of the protein produced in the cells.
Table 6. Effect of media components on the aconitase expression at the harvest time in the E. coli cells
Media
|
Aconitase expression (mg/ g DCW) in BL21(DE3) cells expressing
|
Aconitase
|
Aconitase & GroEL/ES
|
MM
|
22.8
|
23.45
|
LB
|
20.93
|
20.13
|
YT
|
27.61
|
29.04
|
TB
|
35.47
|
37.71
|
Effect of osmolytes/compatible solutes augmented in the media
Osmolytes are known to have a direct impact on the solubility of proteins by assisting in protein folding and preventing protein aggregation. When recombinant E. coli cells were grown in media supplemented with osmolytes (sorbitol, betaine and glutamate), an improved enzymatic activity was observed with all the three osmolytes as compared to controls without the osmolytes (Fig 10A). These experiments demonstrated that the over-expressed protein was able to acquire bio-functionality due to attainment of native structure confirmation in presence of these osmolytes. The presence of glutamate increased aconitase expression by 1.6-fold while also resulting in a 5- fold increase in activity as compared to control, which can be attributed in part to its metabolizable nature.
Effect of osmotic stress in media on recombinant E coli
The E. coli cells when subjected to osmotic stress are triggered to synthesize osmolytes like betaine and trehalose through their cellular metabolism resulting in their accumulation inside the cell in milimolar concentrations [13]. Therefore, in order to induce osmolyte synthesis through osmotic stress, the cells were subjected to increased salinity in the growth media. The recombinant cells previously adapted to grow in osmotic stress exhibited higher growth rates compared to cells inoculated from unadapted cultures. The cells grown under high osmotic stress conditions showed a decrease of ~30% in growth rates and ~60% in biomass yields (Table 7). The osmotic stress conditions induced by increased salinity decreased the aconitase expression while increasing the recombinant aconitase activity. The gradual improvement in the recombinant aconitase activity was observed with increasing osmotic stress conditions. The E. coli cells adapted to grow at higher salinity (0.5M of NaCl) reportedly accumulate high concentrations of betaine and trehalose [14] resulting in improved aconitase activity as compared to control cells grown without any osmotic stress.
Table 7. Effect of osmotic stress in the growth rate, biomass and aconitase yields
Salinity in media
|
Growth rate #
(h-1)
|
Harvest OD600
|
Aconitase
(mg/g DCW)
|
Control
|
0.55
|
2.14 ± 0.05
|
20.93
|
0.1M NaCl
|
0.53
|
1.60 ± 0.02
|
20.75
|
0.2M NaCl
|
0.48
|
1.34 ± 0.04
|
20.17
|
0.3M NaCl
|
0.45
|
0.94 ± 0.09
|
19.53
|
0.4M NaCl
|
0.43
|
0.81 ± 0.02
|
18.74
|
0.5M NaCl
|
0.40
|
0.67 ± 0.01
|
17.57
|
|
|
|
|
|
#The growth rate of the cells in the initial growth phase of the culture
The increment in the aconitase activity was further improved by ~5 folds with the co-expression of GroEL/ES at higher osmotic stress conditions (Fig 10B). While the aconitase activity was improved, there was no significant change in the solubility of recombinant proteins due to osmotic stress in E. coli cells grown at 25°C. This indicates that the increment in the activity of recombinant aconitase is due to the enhancement in the folding of the proteins accumulated as soluble aggregates devoid of functionality due to the presence of the endogenous osmolytes and GroEL/ES.
Effect of pre-induction heat shock on the recombinant aconitase activity in presence of chaperone-assisted folding
The E. coli cells, when subjected to heat shock are induced to produce some constitutive heat shock proteins in order to overcome the heat shock stress. These proteins include some foldases which assist in the folding of mis-folded proteins and proteases which degrade the aggregated moieties. In order to see the effect of pre-induction heat shock on the solubility of aconitase protein, the transformed cells were subjected to a thermal shock by incubating the cultures at elevated temperatures (42OC and 47OC) for 20 minutes before induction. In another set of experiments the cells were subjected to chemically induced heat shock stress by addition of benzyl alcohol in the LB media. This triggers a heat shock like response by fluidizing the cell membranes [15]. The heat shock induced by benzyl alcohol and thermal shock at 42°C resulted in about 2-fold increase in the aconitase activity and thermal shock at 47°C resulted in about 3-fold increase in aconitase activity as compared to control cells grown in LB at 25°C. When cells were subjected to a combination of osmotic stress along with heat stress (chemically induced or thermal shock at 42°C), the aconitase activity increased by 2-fold as compared to cells experiencing only heat shock. The activity was further improved to 3-fold in presence of GroEL/ES. The highest aconitase activity with 4-fold and 5-fold increase in absence and presence of GroEL/ES was obtained with cells subjected to pre-induction heat shock at 47°C indicating that the host cell’s heat shock protein machinery was participating in the folding of recombinant aconitase (Fig 10C). In terms of solubility, cells exposed to benzyl alcohol-induced heat shock had lower solubility than cells exposed to physical heat shock stress at 42°C, while aconitase solubility was not significantly altered when cells were exposed to both physical and chemical heat treatment at 47°C. A profound 7-fold increase in recombinant aconitase activity was observed in cells subjected to a pre-induction heat shock at 47oC and osmotic stress in combination with GroEL/ES chaperone assisted folding.