V. Discussion
5.5 Conclusion
I would like to conclude that the CaENO1 gene can be expressed and secreted in S.
cerevisiae. Furthermore, the CaENO1p tagged with EGFP in frame also can be expressed and displayed fluorescence in the cells, and secreted into the medium. For the truncated proteins, it revealed that all but three of the proteins were detected but most of them also degraded into several smaller size. According to the degradation sizes of the truncated proteins, it seems that the cells would digest the proteins on a specific site in truncated CaENO1-EGFP protein. Although they can be expressed in S.
cerevisiae, none of the truncated CaENO1 fusion protein was detected in medium samples even after concentration. According to the result, only full length CaENO1 can lead the tagged proteins outside the cells. According to the microscope observation, S. cerevisiae cell seems not to recognize the eno-EGFPp as a cell surface binding protein in this study. The enolase encoded by CaENO1 gene was recognized
as secreted protein in S.cerevisiae and exported outside the cell. It suggested the heterologous enolase was undergo the secretion mechanism in S.cerevisiae. The previous report (Elena, L.V., 2006) showed that enolase encoded by ScEno2 can reach the cell surface and described the protein regions involved in its cell surface targeting in S.cerevisiae. Since the protein between C. albicans with S. cerevisiae is similar (ScEno2p and CaEno1p are 76% identities and ScEno1p and CaEno1p are 77%
identities), the conserved region of both enolases maybe are responsible for the targeting. However the result of eno1[1-510]-EGFPp ( N-terminal 170 amino acids of CaEno1p) compared to the N-terminal 169 amino acids residue of ScEno2p did not confirm the the idea that this similar region was also critical to secretion in the CaENO1 gene. Further experiments are needed to determine the mechanism of enolase export in Candida albicans.
VI. Future Work
In this study, only full-length CaENO1 can be detected the secretion in S.cerevisiae. Therefore, there are three issues for the research in the future.
First is the detection system seems not sensitive for the recombinant proteins in this study. Since the CaENO1p was heterologus to S. cerevisiae, when CaENO1 was truncated, it might be not recognized by S. cerevisiae. Therefore, it prefers to identify the secretion signal of CaENO1 in C. albicans. However, the EGFP reporter system contained several CUG codons, and C. albicans translates the standard leucine-CUG codon as serine partially. Therefore the EGFP reporter system cannot be used to the full efficiency in C. albicans, it is need to be changed or modified.
Second is the secretion was not detected within truncated constructs, it might be result from the protein abnormal folding, and therefore the cell would not recognize them as a normal protein. Because the truncated recombinant protein was not stable in the cell, it might indirectly affect the protein secretion. For this problem, one could replace the approach of truncation with random mutagenesis. The random mutagenesis can be used for construction of large and diverse clones to find essential residues of the protein.
Third is that the growth condition might be a factor for the secretion when study in C. albicans. A research by Roger et al. [28] showed that enolase was indentified from cell wall-enriched fraction only in hyphae form C. albicans. It seems that the growth condition (YPD+10 % serum at 37℃ , hyphae indution) might induce the morphic transition and influence the enolase export directly or indirectly, and therefore it is necessary to be considered as a factor for expressing secreted proteins.
VII. Reference
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[15] Prudovsky, I., Mandinova, A., Soldi, R., Bagala C., Graziani, I., Landriscina, M., Tarantini, F., Duarte, M., Bellum, S., Doherty, H. and Maciag, T. (2003) The non-classical export routes: FGF1 and IL-1α point the way. J. Cell Sci. 116, 871–4881.
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Figure 1. Strategy and flow chart of the experiments in this study.
(A)
(B)
Figure 2. Restriction enzyme digestion of YEP363-CaENO1-TP.
(A) The map of YEP363-CaENO1-TP. (B) The result of Nde I digestion.
Lane 1-6 :YEP363–CaENO1-TP plasmid digested by Nde I. Lane 7 : untreated plasmid.
A: 1868 bp DNA fragment. B: 3022bp DNA fragment. C: 5632 bp DNA fragment.
M : 1kb DNA leader.
Figure 3. Western blot of the eno-tp expressed in cell.
Protein sample extracted from S.cerivisiae cell were detected by anti-HA antibody.
Each lane was loaded 10μl of protein sample mixture.
Lane 1-4: protein sample (eno-tp) extracted from 10560-2B-f1 Lane 5 : E-tag protein sample as positive control of western blot.
Lane 6 : protein sample extracted from 10560-2B-f0 contained plasmid YEP363.
A: 55 kDa protein of eno-tp
M : Prestain Protein marker (Cat. No.0901)
Figure 4. Wstern Blot of the eno-tp secreted outside the cell.
Protein sample extracted from cultured medium were detected by anti-HA antibody.
Each lane was loaded 10μl of protein sample mixture.
Lane 1: protein sample as negative control from 10560-2B-f0 Lane 2: E-tag sample as positive control (bands A, B, C).
Lane 3-6 : protein sample from 10560-2B-f1 (band B’)
A: 40 kDa of E-tag. B: 55 kDa of E-tag. B’: 55 kDa of eno-tp. C: 80kDa of E-tag.
M : Protein marker : Prestain Protein marker (Cat. No.0901)
(A)
(B)
Figure 5. The construction of the plasmid expressed CaENO1-EGFP fusion protein in the S. cerivisiae cell.
(A) The strategy of the construction. (B) The PCR product of EGFP on agarose gel. Lane 1 : the PCR product of XbaΙ-EGFP -XbaΙ . Lane 2 : the PCR product of XbaΙ-EGFP-PstΙ. A: 739 bp DNA fragment of XbaΙ-EGFP–XbaΙ. B: 742 bp DNA fragment of XbaΙ-EGFP-PstΙ.
(A)
Figure 6. Restriction digestion of the plasmids expressed CaENO1-EGFP fusion genes.
(A) The maps of the plasmids YEP363-CaENO1-EGFP and YEP363-CaENO1-EGFP-TP. (B) The result of XbaΙ and BsrGI digestion on YEP363-CaENO1-EGFP. Lane 1: the plasmid was digested into 721 bp, 929 bp, 3771 bp and 5694 bp DNA fragments (the arrows showed bands a, b, c, d). (C) The result of XbaΙ and BsrGI digestion on YEP363-CaENO1-EGFP-TP. Lane 1:
the plasmid was digested into 721 bp, 929 bp, 3771 bp and 5830 bp DNA fragments (the arrows showed bands a, b, c, d). M: 1kb DNA leader.
(A) (B)
Figure 7. The western blot of CaENO1-EGFP fusion protein.
The protein samples of cell extract were detected by antibody. Each lane was loaded 10μl of protein sample mixture. (A) the result of western bolt with anti-HA antibody. Lane 1: E-tag sample.( E-tag construct tagged with HAHIS tag was about 40 kDa, but it also can be detected to be about 55 kDa and 80 kDa in this study). Lane 2: the protein sample from 10560-2B-f0.
Lane 3: the protein sample (eno-tp) from 10560-2B-f1. Lane 4: the protein sample (eno-EGFPp) from 10560-2B-f3. Lane 5: the protein sample (eno-EGFP-tp) from 10560-2B-f2. (B) the result of western bolt with anti-EGFP antibody. Lane 1: the protein sample from 10560-2B-f0. Lane 2: the protein sample(eno-EGFPp) from 10560-2B-f3. Lane 3: the protein sample(eno-EGFP-tp)from 10560-2B-f2.
A: 40 kDa protein of E-tag. B: 53 kDa of eno-tp .C and D: 79kDa and 72 kDa of eno-EGFP-tp. E : 75 kDa of eno-EGFPp. F: 79 kDa of eno-EGFP-tp. M : Prestain Protein marker (Cat. No.0901)
(A) (B)
Figure 8. The western blot of supernatant samples of CaENO1-EGFP fusion proteins from the supernatant of cultured medium.
The protein samples of supernatant were detected by antibody. Each lane was loaded 30μl of protein sample mixture. (A) The result of western bolt with anti-HA antibody. Lane 1: E-tag sample. (E-tag construct tagged with HAHIS tag was about 40 kDa, but it also can be detected to be about 55 kDa and 80 kDa in this study). Lane 2: the protein sample from 10560-2B-f0.
Lane 3: the protein sample (eno-tp) from 10560-2B-f1. Lane 4: the protein sample (eno-EGFP-tp) from 10560-2B-f2. (B) The result of western bolt with anti-EGFP antibody.
Lane 1: the protein sample from 10560-2B-f0. Lane 2: the protein sample (eno-EGFP-tp) from 10560-2B-f3. Lane 3: the protein sample (eno-EGFPp) from 10560-2B-f2.
A: 40 kDa of E-tag. B: 53 kDa protein of eno-tp. C: 79kDa protein of eno-EGFP-tp. D: 75 kDa protein of eno-EGFPp . M: Prestain Protein marker (Cat. No.0901)
55KD
A B
95KD
C
72KD 55KD
1 2 3 4
44KD
1 2 3
95KD
72KD
D
(A) 10560-2B-f0
visible light UV light (B) 10560-2B-f2
visible light UV light (C) 10560-2B-f3
visible light UV light
Figure 9. The microscopy observation of CaENO1-EGFP fusion protein.
The transformed cells were incubated for 48 hours and observed under fluorescence microscopy with 400X maganification.
(A) the visible light and UV light of 10560-2B-f0 . (B) the visible light and UV light of 10560-2B-f2 expressing eno-EGFP-tp under uv light. (C) the visible light and UV light of 10560-2B-f3 expressing eno-EGFP under uv light.
To identify the localization and signal region of enolase
Figure 10. The diagram of the strategy in this experiment. To identify the localization and signal region of enolase
CaENO1 signal region
EGFP (reporter)
CaEno1 full length
CaENO1 fragments, truncation by PCR
Negative control
Positive control 1
2 3.
CaENO1-
Truncated fragments
CaENO1-
Full length
EGFP (reporter)
Figure 11. The diagram of the constructs of truncated CaENO1.
The constructs were transformed into S.cerivisiae for analysis of secretion.
1 4.YEP363- CaENO1[388-1320]-EGFP
5.YEP363- CaENO1[451-1320]-EGFP
(A)
(B)
Figure 12. The construction of the plasmid expressing EGFP protein in S. cerivisiae cell.
(A) The strategy of the construction. (B) The PCR product of pACT1 on agarose gel. lane1.
2 : PCR products of pACT1. A: 500 bp DNA fragment of pACT1. M : 1kb DNA marker
The construction – negative control
pACT1 CaENO1 EGFP
YEP363-CaENO1-EGFP
BamHΙ SpeΙ
M
1
2
1 kb 0.75 kb
0.5 kb
A
pACT1 EGFP
BamHΙ SpeΙ
YEP363-EGFP (negative control , With ATG start codon)
(A)
(B)
Figure 13. Restriction digestion of the plasmids expressed EGFP genes.
(A) The maps of the plasmids YEP363 -EGFP (B) The result of BsrGI digestion on plasmid YEP363-EGFP. Lane 1.2: the plasmid was digested into 3643 bp and 6140 bp DNA fragments (band a and b). M: 1kb DNA leader.
6.0kb 4.0kb 5.0kb 3.0kb
M 1 2
a b
(A) (B)
(C) (D)
Figure 14. The construction of the plasmids expressing truncated CaENO1-EGFP protein in S. cerivisiae cell.
(A) The strategy of the construction. (B) The PCR products of truncated CaENO1 on agarose gel. Lane 1: CaENO1/1-150 (150 bp, band A). Lane 2: CaENO1/1-279 (279 bp, band B).
Lane 3: CaENO1/1-387(387 bp, band C). Lane 4: CaENO1/1-510(510 bp, band D). M: 100bp DNA marker (C) The PCR products of truncated CaENO1 on agarose gel. Lane 5: CaENO1 gene (1320 bp, band E). Lane 6: CaENO1/280-1320 (1041 bp, band F). Lane 7:
CaENO1/389-1320 (933 bp, band G). M: 1kb DNA marker (D) The PCR products of truncated CaENO1 on agarose gel. Lane 8: CaENO1/1-451(450bp, band H). Lane 9:
CaENO1/451-900 (450bp, band H’). Lane 10: CaENO1/900-1320 (420 bp, band I). Lane 11:
CaENO1/1-900 (900 bp, band J). Lane 12: CaENO1/451-1320 (869 bp, band K). M: 1kb DNA marker.
YEP363-CaENO1[1-387]-EGFP
(B)
Figure 15. Restriction digestion of the plasmids expressing truncated CaENO1-EGFP fusion gene.
(A) The maps of the plasmids. (B) The result of restriction enzyme digestion.
Lane 1: the plasmid YEP363-CaENO1[1-150]-EGFP was digested by XbaΙ and BsrGI (the arrows showed b, f and g).
Lane 2: the plasmid YEP363-CaENO1[1-279]-EGFP was digested by XbaΙ and BsrGI (the arrows showed d, f and g).
Lane 3: the plasmid YEP363-CaENO1[1-387]-EGFP was digested by XbaΙ and BsrGI (the arrows showed e, f and g).
Lane 4: the plasmid YEP363-CaENO1[1-510]-EGFP by XbaΙ and BsrGI (the arrows showed a, b, f and g).
Lane 5: the plasmid YEP363- CaENO1[280-1320]-EGFP was digested by BsrGI (the arrows showed h, I and j).
Lane 6: the plasmid YEP363- CaENO1[388-1320]-EGFP was digested by BsrGI (the arrows showed h, I and j).
Lane 7: the plasmid .YEP363- CaENO1[1-1450]-EGFP was digested by XbaΙ and BsrGI(the arrows showed l, m, r and s).
Lane 8: the plasmid YEP363-CaENO1[451-900]-EGFP was digested by XbaΙ and BsrGI (the arrows showed n, r and s).
Lane 9: the plasmid YEP363-CaENO1[901-1320]-EGFP was digested by XbaΙ and BsrGI (the arrows showed n, r and s).
Lane 10: the plasmid YEP363-CaENO1[1-900]-EGFP was digested by XbaΙ and BsrGI (the arrows showed n, r and s).
Lane 11: the plasmid YEP363-CaENO1[451-1320]-EGFP was digested by XbaΙ and BsrGI(the arrows showed k, q, r and s).
M: 1kb DNA leader.
a : 397 bp DNA fragment, b: 843 bp and 871 bp, d: 1000 bp, e: 1108 bp, f : 3643 bp, g:
5431 bp, h: 1644bp, i: 3643bp, j: 5550 bp and 5442bp, k: 397 bp, l: 447 bp, m: 774 bp, n:1144 bp, 1174 bp and 1224 bp, q: 1594 bp, r: 643 bp, s:~5431 bp.
A
B C D E F
G
H I J
72KD 55KD 43KD 34KD 26 KD
72 KD 55 KD 43 KD 34 KD 26 KD
P 1 2 3 4 5 6 7 N
P N 8 9 10
Figure 16. The detection of truncated CaENO1-EGFP protein in S. cerivisiae cell with western blot analysis.
The transformed cells were incubated for 48 hours and broken to extract the protein.
Then the protein samples were analyzed by anti-EGFP antibody by western blot analysis. Each lane was loaded 10μl of protein sample mixture.
N (negative control) : the protein sample from 10560-2B-f0
P (positive control) : the protein sample (eno-EGEPp) from 10560-2B-f3.
truncated CaENO1-EGFP fusion protein samples :
1.eno1[1-150-]EGFP p (band A) 2.eno1[1-279]-EGFP p (band B) 3.eno1[1-387]-EGFP p (band C) 4. eno1[1-450]-EGFP p (band D)
5.eno1[1-510]-EGFP p (band E) 6.eno1[1-900]-EGFP p (band F) 7. EGFP (band G) 8.eno1[280-1320]-EGFPp(band H) 9.eno1[389-1320]-EGFPp(band I)
10.eno1[451-1320]-EGFPp(band J) 11.eno[451-900]-EGFPp (band K) 12. eno[901-1320]-EGFPp (band L)
A: ~31kDa protein B : ~43kDa C: ~40kDa D: ~43kDa E: ~45kDa F: ~59kDa kDa G:
~26kDa H: ~65kDa I: ~62kDa J: ~58kDa K: ~34kDa L:~34kDa M: Prestain Protein marker (Cat. No.0901)
K L
11 12
72 KD 55 KD 43 KD 34 KD 26 KD
1.10560-2B.- EGFP
2. 10560-2B-CaENO1[1-150]
3.10560-2B-CaENO1[1-279]
4.10560-2B-CaENO1[1-387]
5.10560-2B-CaENO1[1-450]
6.10560-2B-CaENO1[1-510]
7.10560-2B-CaENO1[1-900]
8.10560-2B-CaENO1[280-1320]
9.10560-2B-CaENO1[388-1320]
10.10560-2B-CaENO1[451-1320]
11.10560-2B-CaENO1[901-1320]
12.10560-2B-CaENO1[451-900]
Figure 17. The microscopy observation of truncated CaENO1-EGFP proteins in
S. cerivisiae cells.
S. cerivisiae cells were incubated for 48 hours and observed under fluorescence microscopy with 400X magnification.
8.10560-2B-f0
Figure 18. The western blot of truncated CaENO1-EGFP fusion proteins from cultured medium.
The protein samples of supernatant were detected by anti-EGFP antibody with western blot analysis. Each lane was loaded 30μl of protein sample mixture.
Western blot of truncated CaENO1-EGFP proteins.
N: protein sample as negative control, extracted from 10560-2B-f0
P: positive control, eno-EGFPp sample extracted from cells of 10560-2B-f3.
1.eno1[1-150-]EGFP p (~31kDa) 2.eno1[1-279]-EGFP p (~36kDa) 3.eno1[1-387]-EGFP p (~40kDa) 4. eno1[1-450]-EGFP p (~43kDa)
5.eno1[1-510]-EGFP p (~45kDa) 6.eno1[1-900]-EGFP p (~59kDa) 7.EGFP (~26kDa) 8.eno-EGFPp(~75kDa) 9.eno1[280-1320]-EGFPp(~65kDa).
10.eno1[389-1320]-EGFPp (~62kDa) 11.eno1[451-1320]-EGFPp (~58kDa).
12.eno[451-900] (~43kDa) 13.eno[901-1320] (~43kDa) . A: ~75 kDa protein; M: Prestain Protein marker (Cat. No.0901)
P 1 2 3 4
P 9 10 11 12
5 6 7 8 P 13 N P
A
Figure 19. The western blot of concentrated supernatant.
The 5 ml supernatants of cultured media for carrying truncated CaENO1-EGFP fusion gene were concentrated to 250μl and analyzed by anti-EGFP antibody with western blot analysis. Each lane was loaded 30μl of protein sample mixture.
P : eno-EGFPp protein sample extracted from cells.
1. eno-EGFPp (~75kDa)
2. protein sample as negative control from 10560-2B-f0 3. EGFP (~26kDa)
4. eno1[1-450]-EGFP p (~43kDa) 5. eno[451-900]-EGFPp (~43kDa) 6. eno[901-1320]-EGFPp(~43kDa) 7.eno1[1-900]-EGFPp(~59kDa) 8. eno1[451-1320]-EGFPp(~58kDa) A: ~75 kDa protein
M: Prestain Protein marker (Cat. No.0901)
1 2 3 4 P
72kD 55kD
72kD 55kD
P 5 6 7 8
34kD 26kD
34kD 26kD
A
Figure 20. The western blot of concentrated supernatants.
The 50 ml supernatants of cultured media for truncated CaENO1-EGFP fusion gene were concentrated to 250 μl and analyzed by anti-EGFP antibody with western blot analysis. Each lane was loaded 30μl of protein sample mixture.
P : eno-EGFPp sample extracted from cell.
1. eno-EGFPp (~75kDa).
2. protein sample as negative control, extracted from 10560-2B-f0.
3. EGFP (~26kDa).
4. eno1[1-450]-EGFP p (~43kDa) 5. eno[451-900]-EGFPp (~43kDa).
6. eno[901-1320]-EGFPp(~43kDa) 7. eno1[1-900]-EGFPp (~59kDa).
8. eno1[451-1320]-EGFPp(~58kDa) M: Prestain Protein marker (Cat. No.0901)
N M 1 2 3
5 6 7 8 P 4 P
72KD 55KD 34KD 26KD
55KD 72KD
34KD 26KD
(A)
(B)
C: MERGE D: DIC
D: DIC B: EGFP A: Alexa Fluor 594 WGA
C: MERGE
A:Calcofluor white B:EGFP
Figure 21. The localization of CaENO1p in S. cerevisiae cell.
The S. cerevisiae cells were stained with Alexa Fluor 594 WGA (which binds to sialic acid and N-acetylglucosaminyl residues) for cell membrane labeling and Calcofluor white (which binds to beta-1,3 and beta-1,4 polysaccharides ) for cell wall labeling.
The treated cell was fixed on slide to observe under confocal microscope with 1000X magnification.
(A) The transformed cell (10560-2B-f2) stained with Calcofluor white (blue )for cell wall labeling .
(B) The transformed cell (10560-2B-f2) stained with Alexa Fluor 594 WGA (red) for cell membrane labeling.
A: Dye of Alexa Fluor 594 WGA. B: Dye of EGFP. C: A and B merged. D: image of DIC (Differential Interference Contrast)
(A)
(B)
D: DIC
A: Alexa Fluor 594 WGA B: EGFP
C: MERGE D: DIC
C: MERGE
A: Calcofluor white B: EGFP
Figure 22. The localization of EGFP in S. cerevisiae cell.
The S. cerevisiae cells were stained with Alexa Fluor 594 WGA (which binds to sialic acid and N-acetylglucosaminyl residues) for cell membrane labeling and Calcofluor white (which binds to beta-1,3 and beta-1,4 polysaccharides ) for cell wall labeling.
The treated cell was fixed on slide to observe under confocal microscope with 1000X magnification.
(A) The transformed cell (10560-2B- EGFP) stained with Calcofluor white (blue) for cell wall labeling .
(B) The transformed cell (10560-2B- EGFP) stained with Alexa Fluor 594 WGA (red) for cell membrane labeling.
A: Dye of Alexa Fluor 594 WGA. B: Dye of EGFP. C: A and B merged. D: image of DIC (Differential Interference Contrast)
(A)
(B)
Figure 23. The blast result of enolase between S.C. and C.A.
(A) Query: CaEno1p, sbjct : ScEno2p (B) Query: CaEno1p, sbjct : ScEno1p
93
50
129 170
簡歷
許淑貞
Shiu, Shu - Jen
主要求學歷程:2006-2008 國立交通大學
生物科技學院 生化工程所
2002-2006 國立高雄大學 生命科學系
1999-2002 私立永年高級中學