建立高產量分泌重組蛋白的模式於嗜甲醇酵母菌

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研究生：	劉錫輝 Shi-Hwei Liu
論文名稱：	建立高產量分泌重組蛋白的模式於嗜甲醇酵母菌 Process model for high-level secretory production of recombinant protein in Pichia pastoris
指導教授：	張大慈 Margaret Dah-Tsyr Chang
口試委員:
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生命科學系 Department of Life Sciences
論文出版年：	2005
畢業學年度：	94
語文別：	英文
論文頁數：	88
中文關鍵詞：	重組蛋白、蛋白質分泌系統、糖化酵素、嗜甲醇酵母菌
相關次數：	點閱：3 下載：0
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雖然嗜甲醇酵母菌 (Pichia pastoris) 已經發展成為廣泛使用的宿主微生物生產大量的重組蛋白，我們的研究發現欲將根瘤菌的糖化酵素 (glucoamylase) 分泌於嗜甲醇酵母菌中遭遇到很大的瓶頸。在此論文中，我們利用遺傳工程的方式設計一段改造過的前導序列 (modified signal peptide) 代替糖化酵素的前導序列，並增加此段前導序列替換後糖化酵素的基因數目，接著再共同表現一段SEC4基因，最後結合這些有利因子使糖化酵素的分泌表現提升了一百倍。SEC4基因掌控的蛋白質屬於Rab家族的GTPase，它能插入分泌的小泡中，為蛋白質分泌的過程中不可或缺的角色。為了更進一步了解SEC4在嗜甲醇酵母菌中的生理角色，我們在已經插入七個糖化酵素基因數目的嗜甲醇酵母菌中表現大量的定點突變蛋白SEC4S28N，並且也構築一組含不同的基因數目之野生型SEC4之選殖株。我們發現此定點突變會減低SEC4蛋白和GTP結合的親合力阻止分泌小泡傳送和融合至細胞膜，進而抑制細胞生長和糖化酵素的分泌表現。我們的研究也顯示，經由酒精氧化酶的啟動子 (AOX1 promoter) 啟動大量正常SEC4 基因表現會增加糖化酵素在高濃度的細胞中之分泌量，但卻會抑制酵母菌細胞的生長，並隨著基因量的增加而更形嚴重。有趣的是，表現單一的SEC4 基因會在較早的培養時期造成細胞生長減慢，但卻會維持較高的未水解SEC4 蛋白至培養的後期，顯示在嗜甲醇酵母菌中維持定量的SEC4 蛋白並不會影響到細胞的生長。根據此觀點，我們將表現SEC4 蛋白的酒精氧化酶啟動子置換成甘油醛-3-磷酸去氫酶的啟動子 (GAP promoter) 發現糖化酵素的分泌仍能提升，但卻不會影響細胞的生長。綜合而言，SEC4 基因及SEC4 蛋白劑量能調節嗜甲醇酵母菌的生長和重組蛋白的胞外分泌表現。

The methylotrophic yeast Pichia pastoris has been developed as a widely used host organism for high-level production of recombinant protein. However, we found that the secretion of Rhizopus oryzae glucoamylase (GA) encountered certain bottleneck in P. pastoris. In this study, R. oryzae GA was genetically engineered with a modified signal peptide (MSP), increased copy number of the gene, and coexpression of SEC4, a gene encoding a Rab protein associated with secretory vesicles, and it is to achieve a secretion level up to 100-fold in P. pastoris. Subsequently, to elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4S28N, was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4S28N mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4S28N gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated a delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. The expression of only one copy of SEC4 resulted in delay of cell growth at an early stage, while still maintained high level Sec4p after long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but no inhibition of cell growth were achieved. Taken together, our results demonstrate that SEC4 is substantial for regulating cell growth and heterologous protein secretion in P. pastoris.

Abstract in Chinese……………………………………………………………………..…...1
Abstract…..………………………...………………………………………………………...    3
Abbreviations……………………..…………………………..………………….…...……...5
Chapter 1 Background……………………………………………………………....……....6
1-1.     The methylotrophic yeast Pichia pastoris...…………………………….…….6
1-2.     The P. pastoris expression system………………………………..…………...7
1-3.         Methanol utilization phenotypes…………………………………………........8
1-4.         Post-translational secretory pathway of P. pastoris…………………………...8
Chapter 2 Improved Secretory Production of Glucoamylase in Pichia pastoris
by Combination of Genetic Manipulations……………………………………11
2-1.     Introduction……………………………………………………….……….....11
2-2.     Materials and methods……………………………………………….…..…    ..14
2-2-1.     Strains, vectors and reagents………………………………………………    ....14
2-2-2.     Construction of recombinant plasmids encoding glucoamylase……………..14
2-2-3.     Transformation of P. pastoris………………………………………………    ..15
2-2-4.     Identification of P. pastoris transformants containing multiple copy
    number of MSPGA genes…………………………………………………….16
2-2-5.     Isolation of genomic DNA…………………………………………….……    ..16
2-2-6.     Southern blotting……………………………………………………………..17
2-2-7.     Expression of GA in P. pastoris……………………………………………...18
2-2-8.     SDS–PAGE and Western blotting…………………………………….……...19
2-2-9.     GA activity assay……………………………………………………...……    ..19
2-2-10.     Integration of SEC4 in each MSPGA/P. pastoris transformant………..….....20
2-2-11.     Purification of recombinant GA……………………………………...……....20
2-3.     Results……………………………………………………………...………...22
2-3-1.     Generation of recombinant P. pastoris containing a single copy
    insert of WTSPGA and MSPGA……………………………………………...22
2-3-2.     Analysis of GA secretion in WTSPGA-1 and MSPGA-1…………..…….....23
2-3-3.     Isolation of recombinant P. pastoris with multi-copy MSPGA gene…….…..23
2-3-4.     Enhancement of GA secretion by overexpression of SEC4 in each
    MSPGA/P. pastoris transformant…………………………………………….25
2-4.     Discussion……………………………………………………………..……    ..26
Chapter 3 Molecular Genetic Manipulation of Pichia pastoris SEC4 Governs
Cell Growth and Glucoamylase Secretion……………………………….…....36
3-1.     Introduction……………………………...…………………………………...36
3-2.     Materials and methods……………………………………………………….39
3-2-1.     Strains, vectors and reagents…………………………………………………39
3-2-2.     Construction of a dominant-negative mutant of SEC4 and a set of
    multi-copy wild type SEC4 expression cassette in
    P. pastoris MSPGA-7………………………………………………………..39
3-2-3.     Southern blotting……………………………………………………………..41
3-2-4.     Overexpression of wild type and mutant SEC4 in MSPGA-7 on
    culture plates……………………………..…………………………………..42
3-2-5.     Growth conditions and preparation of recombinant yeast cells……………...42
3-2-6.     Identification of recombinant Sec4p………………………………………....43
3-2-7.     Analysis of glucoamylase (GA) secretion and intracellular GA…….……….43
3-3.     Results……………………………………………………………………......45
3-3-1.     Construction of SEC4S28N in P. pastoris strain MSPGA-7…………………...45
3-3-2.     Growth effects of SEC4S28N overexpression in P. pastoris MSPGA-7…….....46
3-3-3.     The delay of growth and overexpression of Sec4p parallel copy number of
    SEC4 in MSPGA-7…………………………………………………………...48
3-3-4.     Effects on GA secretion by overexpression of SEC4S28N and a set of
    multi-copy of SEC4 in P. pastoris MSPGA-7……………………….……….49
3-3-5.     Analysis of intracellular GA in P. pastoris transformants……………………50
3-3-6.     Analysis of GAP promoter-drived SEC4 on GA secretion in MSPGA-7……51
3-4.     Discussion……………………………………………………………………52
Chapter 4 Conclusion and Discussion……...…………………………………….………..69
References…………………………………………………………………………….……..72
Appendix………………………………………………………………………...…………..82

List of Tables
Table 1 Determination of copy number of MSPGA inserts in recombinant P. pastoris……    34
Table 2 Comparison of GA production in P. pastoris transformants………………….……    35
Table 3 Summary of cell growth and GA production in P. pastoris transformants…...……    68

List of Figures
Figure 1. The diagrams of WTSPGA and MSPGA…………………………………...……...29
Figure 2. Determination of copy number in P. pastoris transformants harboring WTSPGA
and MSPGA inserts…………………………………………………………………30
Figure 3. Comparison of GA secretion by WTSPGA-1 and MSPGA-1……………………    ..31
Figure 4. Enhancement of GA secretion in P. pastoris transformants containing different
copy number of MSPGA inserts……………………………………………………32
Figure 5. Overexpression of SEC4 improved secretory production of GA…………...……...33
Figure 6. Determination of copy number of SEC4 and SEC4S28N inserts in
P. pastoris MSPGA-7……………………………………………………………....56
Figure 7. Effects on growth of different P. pastoris transformants upon
overexpression of SEC4 and SEC4S28N…………………………………………...57
Figure 8. Time-dependent growth of P. pastoris transformants were assayed………..……...58
Figure 9. Western blotting analysis of mutant and wild type Sec4p………………….……...59
Figure 10. Comparison of Sec4p in each clone cultured in BMM for 24 h and 120 h………60
Figure 11. Growth effects on overexpression of a set of multi-copy SEC4 in
P. pastoris MSPGA-7……………….…………………………………………....61
Figure 12. Time-dependent growth of P. pastoris transformants were assayed……………...62
Figure 13. The amounts of intrcellular Sec4p in P. pastoris transformants were
detected by Western blotting……………………………………………………..63
Figure 14. Effect of overexpression of mutant and wild type SEC4 on GA secretion
in P. pastoris MSPGA-7………………………………………………………….64
Figure 15. Quantitative analysis of intracellular GA by Western blotting…………………...65
Figure 16. Time-dependent production of intracellular GA in P. pastoris
transformants containing a set of different copy number of SEC4………………66
Figure 17. SDS-PAGE analysis of GA secretion in P. pastoris transformants……………….67

                                

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