6. Heterologous overexpression of the hyperthermophilic putative amino acid transporter
6.2. Selection of suitable culture conditions and the optimal expression strain for AAT
strain YpAAT were submitted to different conditions of growth and induction of gene expression.
For each transformant, starter cultures were first established. They grew for 48h, until an OD600
pITy-QC
ura3d
2µ ORI δsequences
XhoI
His6
XhoI AAT tCYC1 NeoR ORI pGAL1
The strains were then inoculated to an OD600 of 0,02 at 30 C into a volume of 200 ml of non-inducing selective medium containing raffinose, or rich medium containing glucose, as the sole carbon source (for description of the media composition, see the Material and methods section, chapter 13.2.1).
From the 200 ml uninduced culture, 50 ml each were withdrawn at four different growth phases., i.e., at OD600 of 1 to 2, 4 to 5, 7 to 9, and in the stationary phase (indicated by arrows in Fig. 36). The growth curves in selective medium are shown in Fig. 36 (growth curves in rich medium presented no significant differences). The aliquots were then supplemented with 2% galactose to induce the expression of Aatp. After 4h of induction, 25 ml were collected from each culture, cells harvested and frozen. The remaining 25 ml were induced for a total of 8h before being harvested and frozen as well.
The same procedure was performed in selective and rich medium for each transformant.
Fig. 36: Growth curves of different YpAAT transformants in non-inducing selective medium. Data were obtained from three different transformants, #1, #2 and #3. Arrows indicate the timepoints where samples were collected to be analysed as described above (OD600
of 1 to 2, 4 to 5, 7 to 9, and ~12 in the stationnary phase).
The protocol for the determination of the optimal growth and induction conditions is summarized in Fig. 37.
Growth curves of the yeast culture for different YpAAT transformants
0 5 10 15
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Time (hours)
OD 600 nm
#1
#2
#3
Fig. 37: Protocol followed to determine the optimal growth conditions and concurrent high level Aatp expression.
After the induction, yeast membranes of each yeast cells samples were isolated, and the expression level of Aatp was analysed for each of them by immunoblotting, with an anti-His antibody directed towards the His6-tag of Aatp. During the isolation of yeast membranes, extrinsic proteins were removed by a stripping-step, to enrich the sample in Aatp. This was performed by adding 400 mM NaCl to the homogenisation buffer.
We first compared the Aatp expression level in the membranes of yeast cells grown in rich medium, at different OD600 and after different induction times. From the Western-blot in Fig. 38A, it is clear that, in rich medium, the highest Aatp expression level was obtained when the induction was started at an OD600 of 6 to 9 and performed for 8h. Densitometric analysis of the Western blot signals yielded
Transformant #1 Transformant #2 Transformant #3 Identical procedure
as for transformant #1
Starter culture 48h growth
OD600= 11 200 ml yeast culture
50 ml 50 ml 50 ml 50 ml OD600=1 to 2 OD600=4 to 5 OD600=7 to 9 OD600=11
Induction with galactose for different times
25 ml 25 ml 25 ml 25 ml 4h
8h
4h 8h
4h 8h
4h 8h
25 ml 25 ml 25 ml
Isolation of yeast membranes and quantification of the expression level of Aatp by immunoblot
Expression culture in selective medium ScRaff/Gal
Expression culture in rich medium YPD/YPGal Identical procedure as for
the expression culture in selective medium
25 ml
Fig. 38:Overexpression of Aatp under different growth conditions. Aliquots were collected from the cultures at different OD600 (2, 5, 6, 7, 9, 11), and induced for 4h or 8h with galactose. Ten µg of isolated total yeast membranes were analysed by Western-blot or dot-blot. 38A and 38B: Western-blot and corresponding quantification graph showing the amount of expressed Aatp at different times of growth in rich medium, after different times of galactose induction. Arrows indicate the best expression levels. 38C and 38D: Dot-blot and corresponding quantification graph showing the amount of expressed Aatp at different times of growth in selective medium, after different times of galactose induction. The arrow indicates the best expression level.
The Dot-blot of Aatp expression in selective medium in Fig. 38C shows clearly that the best Aatp expression level was obtained when the cells were at an OD600 of 5 at the induction start and protein expression was induced for 8h. Densitometric analysis of the Dot blot signal yielded a relative intensity of ~7000 as pointed out on the corresponding quantification graph Fig. 398D).
The best expression levels in the two different media were then directly compared on the same Western blot. (Fig. 39A). The blot and its corresponding quantification graph (Fig. 39B) show clearly that Aatp was better expressed in cells grown in selective medium than in rich medium, yielding 4500 and 9000 relative units, respectively.
The expression level of Aatp was also checked in the YpMEGA/AAT strain that carries the pMEGA vector for enhanced AAT expression from the GAL1 promoter. As for Ste2mp, it was expected that the constitutive overexpression of the Gal transcription factors from the pMEGA vector would lead to an increased transcription of the AAT gene and, consequently, to a higher Aatp expression level.
The Western blot with total membranes isolated from YpMEGA/AAT that had been grown and induced under the same conditions as the YpAAT shows that Aatp expression is not boosted by coexpression of the GAL transcription factors (Fig. 39C).
OD 6 OD 9 OD 11 4h 8h 4h 8h 4h 8h
38A OD 2 OD 5 OD 7 OD 11
4h 8h 38C
Expression level of AAT in selective medium
0 2000 4000 6000 8000 relativ densitometry
4h 8h 4h 8h 4h 8h 4h 8h OD 2 OD 5 OD 7 OD11 38D
relative intensity
Relative quantification of the amount of expressed Aatp in selective medium
under different conditions Expression of AAT in rich medium
0 2000 4000 6000 8000 10000 38B
4h 8h 4h 8h 4h 8h OD 6 OD 9 OD 11 Relative
intensity
Relative quantification of the amount of expressed Aatp in rich medium
under different conditions
The quantification graph of the densitometric analysis of the Western-blot signals (Fig. 39D) indicates a considerably lower intensity for Aatp expressed in the YpMEGA/AAT strain (~2000 units) than in YpAAT (~6500 units).
Fig. 39: Comparison of the Aatp expression levels in cells grown in two different media and between different strains. Comparison from same amounts of total membranes (20 µg). 39A and 39B: Comparison between cells giving the best level of expression in each medium in the strain YpAAT: in rich medium: OD600 of 6, induction 8h; in selective complete medium: OD600 of 4, induction 8h. 39A: Western-blot showing the expressed Aatp in cells grown in rich medium or selective complete medium. 39B: Corresponding quantification graph comparing the amount of expressed Aatp in the two different media. 39C: Western-blot showing the expressed Aatp in the YpAAT strain and in the YpMEGA/AAT strain. 39D: Corresponding quantification graph comparing the amount of expressed Aatp in the two different strains.
From the results outlined above, we chose to express Aatp in YpAAT induced in selective medium at an OD600 between 4 to 5 and to induce gene expression with galactose for 8h.
Rich selective negative medium medium control 39A
Comparison of the expression level of Aatp in cells grown in different media
0 2000 4000 6000 8000 10000 relativ e intensity
39B
rich selective negative medium medium control
YpAAT YpMEGA/AAT
strain strain 39C
Comparison of the expression level of AAT in different strains
0 2000 4000 6000 8000 relative intensity
39D
YpAAT YpMEGA/AAT sstrain strain