人類顆粒細胞增生因子（Human Granulocyte Colony Stimulating Factor；hG-CSF）能刺激嗜中性白血球前驅細胞生長，並促進其增殖、分化，同時具有促進嗜中性白血球由骨髓釋出及增強成熟嗜中性白血球的機能，它被廣泛的應用在癌症化療和骨髓移植後，所引起的嗜中性白血球缺乏症（neutropenia）。
酵母菌Pichia pastoris是常用於表現重組蛋白的宿主細胞之一，具有將蛋白質正確地折疊、轉譯後修飾以及將蛋白分泌至胞外等優點。在我們實驗室利用發酵槽誘導酵母菌可以獲得hG-CSF約71 mg/L。
對宿主細胞而言的密碼使用頻率較高的基因可能會使蛋白產量有所提昇，因此本實驗依據酵母菌的密碼偏好，設計並合成出最佳化密碼的hG-CSF基因（syn hG-CSF），利用分析CAI值預測此段基因在大腸桿菌E. coli和酵母菌P. pastoris之中會有較高的蛋白表現，之後分別轉殖到大腸桿菌BL21（DE3）跟酵母菌SMD1168之中。
目前已成功獲得大腸桿菌轉型株，在37℃下，使用搖瓶的方式加入IPTG誘導4個小時表現syn hG-CSF，以SDS-PAGE及西方點墨法（Western blotting）分析，發現所表現的蛋白分子量符合在22 kDa的位置，在500毫升的菌液當中大約有100毫克目標蛋白。

The recombinant human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that has been widely used for the treatment of neutropenia after chemotherapy and bone marrow transplantation. It can stimulate both proliferation and differentiation of neutrophils.
Pichia pastoris has been extensively used as a cellular host for recombinant protein expression. It’s advantages are posttranslational modification, protein folding correction and protein secretion to the medium. In our laboratory, the expression of hG-CSF in P.pastoris,about 71 mg/L of recombinant protein in bioreactor.
In theory, the higer the codon usage bias by th host cell, the more improved yield of protein.Therefore in this study, we designed and synthesized a new hG-CSF gene by using yeast preferred codon.The use of CAI values predicted this gene would yield  higher expression level when transferred into E. coli BL21(DE3) and P.pastoris SMD1168.
Up to the present, we have successfully cultured the colony of E. coli. after 4 hours of  induction by IPTG at 37℃, the expression of the recombinant protein was confirmed by SDS-PAGE and Western blotting. The amount of recombinant protein was 100 mg in 500 ml LB medium approximately, and molecular weight was as predicted at 22 kDa corresponding to the commercial standard.

封面內頁	
口試委員審議通過簽名表	
授權書	
致謝	
中文摘要	I
英文摘要	II
目錄	III
圖表目錄	V
	
第一章　緒論	
第一節　前言	1
第二節　人類顆粒細胞增生因子	2
第三節　生物細胞內蛋白質合成之密碼使用偏好	5
第四節  原核細胞表現系統	7
	
第二章　實驗材料、設備	
第一節　實驗材料	14
第二節　實驗設備	17
第三節　培養基配置	18
	
第三章　實驗方法	
第一節　hG-CSF synthesize gene基因選殖	20
第二節　pET25b表現質體和syn hG-CSF基因之重組	33
第三節　基因表現	37
第四節　重組蛋白質之純化	43
	
第四章　實驗結果	
第一節　基因建構	45
第二節　確認和純化大腸桿菌所表現之重組蛋白	47
	
第五章　結論與未來展望	
第一節  實驗結論	63
第二節  未來展望	64
	
第六章　參考資料	69
	
	
表 1.1	Triplet codon與胺基酸的對應表	13
		
表3.1	PCR（gene extension）反應之各項成分	24
表3.2	PCR（gene amplification）反應之各項成分	24
表3.3	PCR（gene extension）反應條件	25
表3.4	PCR（gene amplification）反應條件	25
表3.5	TA ligation之各項成分	29
表3.6	pGEM::hG-CSF DNA以限制酶（NcoI、NotI）水解反應組成	32
表3.7	pET25b 質體DNA以限制酶(NcoI、NotI)水解之反應組成。	33
表3.8	pGEM::syn hG-CSF以限制酶（NcoI、NotI）水解之反應組成。	34
表3.9	接合反應溶液。	35
表3.10	pET25b::syn hG-CSF以限制酶（NcoI、NotI）水解之反應組成。	36
		
表5.1	生物技術工業常用的蛋白質表現系統	67
		
圖 1.1	不同增生因子對造血幹細胞分化圖	9
圖 1.2	G-CSF蛋白質分子之三級結構	10
圖 1.3	hG-CSF與其他相似結構之細胞激素比較圖	11
圖 1.4	原核細胞表現系統蛋白質調控機制	12
		
圖 2.1	pET25b(+)表現載體	19
		
圖 3.1	以酵母菌的codon usage所擬出之hG-CSF DNA序列及其對應胺基酸	20
圖 3.2	依照所擬的hG-CSF DNA序列來設計之primer位置圖	22
圖 3.3	PCR反應說明圖	23
圖 3.4	TA vector：pGEM®-T Vector質體圖	28
圖 3.5	轉漬槽示意圖	42
		
圖 4.1	hG-CSF（上） 與syn hG-CSF（下）DNA序列比對圖	50
圖 4.2	hG-CSF（上）與syn hG-CSF （下）DNA轉譯出的胺基酸序列圖	51
圖 4.3	a) hG-CSF
(b) syn hG-CSF在大腸桿菌中的CAI值	52
圖 4.4	洋菜膠電泳分析PCR（gene extension）之結果	53
圖 4.5	洋菜膠電泳分析PCR（gene amplification）之結果	54
圖 4.6	TA cloning 藍白篩選	55
圖 4.7	洋菜膠電泳分析pGEM::syn hG-CSF DNA	56
圖 4.8	(a)洋菜膠電泳確認pET25b::syn hG-CSF DNA
(b)pET25b::syn hG-CSF質體圖	57
圖 4.9	誘導重組蛋白表現	58
圖 4.10	誘導時間和誘導蛋白表現量的關係圖	59
圖 4.11	破菌前後之SDS-PAGE 和 Western blotting	60
圖 4.12	利用市售hG-CSF進行蛋白質定量	61
圖 4.13	純化蛋白（inclusion body）之SDS-PAGE 和 Western blotting	62
		
圖5.1	異源蛋白質分泌至胞外示意圖	68

巫凱隆。2005。利用大腸桿菌系統表現人類白血球增生因子及定性分析。東吳大學化學研究所碩士班論文。

Bahrami, A., Shojaosadati, S.A., Khalizadeh, R., Mohammadian, J., Farahani, E.V., and Masoumian, M.R. (2009) Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives. Biotechnol. Appl. Biochem. 52, 141-148.

Brinkmann, U.,Mattes, R.E.,and Buckel, P. (1989) Hig-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene. 85, 109-114

Brocca, S., Schmidt-Dannert, C., Lotti, M., Alberghinaand, L., and  Schmid, R.D. (1998) Design, total synthesis, functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase. Protein Sci. 7, 1415-1422.

Chung, B.H., Sohn, M.J., Oh, S.W., Park, U.S., Poo, H., Kim, B.S., Yu, M.J., and Lee, Y.I. (1998) Overproduction of Human Granulocyte-Colony Stimulating Factor Fused to the pelB Signal Peptide in Escherichia coli. Journal of Fermentation and Bioengineering. 85, 443-446.

Francis, Y.M., and Graham, S. (2002) Synonymous codon usage bias and expression of human glucocerebrosidase in the methlotrophic yeast,Pichia pastoris. Protein Expression and Purification 26, 96-105

Hartner, F.S., and Glieder, A. (2006) Regulation of methanol utilisation pathway genes in yeasts. Microb. Cell Fact. 5, 2839-2859.

Hill, C. P., Osslund, T. D., and Eisenberg, D. (1993) The structure of granulocyte-colony-stimulating factor and its relationship to other growth factor. Proc. Natl. Acad. Sci. USA. 90, 5167-5171.

Hoglund, M. (1998) Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)-What is the difference? Med Oncol. 15, 229-233

Ikemura, T. (1985) Codon usage and tRNA content in unicellular and multicellular organisms. Mol. Biol. Evol. 2, 13-34.
King, J.L., and Jukes, T.H. (1969) Non-Darwinian evolution. Science 164, 788-798
Metcalf, D. (1985) The granulocyte-macrophage colony stimulating factors. Cell. 43, 5-6.
Metcalf, D. (1990) The colony-stimulating factors: discovery, development and clinical application. Cancer. 65, 2185-2195.

Lasnik, MA., Porekar, VG., and Stalc, A. (2001) Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris. Pflugers Arch - Eur. J. Physiol. 442, 184-186.

Li, Z.,Homg, G.,Wu, Z.Hu, B.,Xu, J.,and Li, L. (2008) Optimization of the expression of hepatitis B virus e gene in Pichia pastoris and immunological characterization of the product. Journal of Biotechnology. 138, 1-8

Lu, H. S., Clogston, C. L., Narhi, L. O., Merewether, L. A., Pearl, W. R., and Boone, T. C. (1992) Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. J. Biol. Chem. 267, 8770-8777.

Mohsen, A.W., and Vockley, J. (1995) High-level expression of an altered cDNA encoding human isovaleryl-CoA dehydrogenase in Escherichia coli. Gene. 160, 263-267
Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J .Immunol Methods. 65, 55-63.
Nagata, S., Tsuchiya, M., Asano, S., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., and Yamasaki, T. (1986) The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor. EMBO J. 5, 575-581.
Nicola, N.A., Metcalf, D., Matsumoto, M., and Johnson, G.R. (1983) Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor. J. Biol. Chem. 258, 9017-9023.
Nomura, H., Imazeki, I., Oheda, M., Kubota, N., Tamura, M., Ono, M., Ueyama, Y., and Asano, S. (1986) Purification and characterization of human granulocyte colony-stimulating factor (G-CSF). EMBO J. 5, 871-876.
Ostergaard, S., Olsson, L., and Nielsen, J. (2000) Metabolic engineering of Saccharomyces cerevisiae. MMBR. 64, 34-50.
Outchkourov, N.S., Stiekema, W.J., and Jongsma, M.A. (2002) Optimization of the expression of equistatin in Pichia pastoris. Protein Expr. Purif. 24, 18-24.
Rangwala, S.H., Finn, R.F., Smith, C.E., Berberch, S.A., Salsgiver, W.J., Stallings, W.C., Glover, G.I., and Olins, P.O. (1992) High-level production of active HIV-1 protease in Escherichia coli. Gene. 122, 263-269
Sharp, P.M., and Li, W.H. (1987) The codon adaptation index – a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 15, 1281-1295.
Shyu, W.C., Lin, S.Z., and Yang, H.I. (2004) Functional Recovery of Stroke Rats Induced by Granulocyte Colony-Stimulating Factor–Stimulated Stem Cells. Circulation. 110, 1847-1854.
Sinclair, G., and Choy, F.Y.M. (2002) Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast Pichia pastoris. Protein Expr. Purif. 26 , 96-105.
Socolovsky, M., Lodish, H. F., and Daley, G.Q. (1998) Control of hematopoietic differentiation: lack of specificity in signaling by cytokine receptors. Proc. Natl. Acad. Sci. USA. 95, 6573-6575.
Souza, L.M., Boone, T.C., Gabrilove, J., Lai, P.H., Zsebo, K.M., Murdock, D.C., Chazin, V.R., Bruszewski, J., Lu, H., and Chen, K.K. (1986) Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232, 61-65.
Stemmer, W.P.C., Crameri, A., Ha, K.D., Brennan, T.M., and Heyneker, H.H. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodexyribonucleotides. Gene. 164, 49-53

Studier, F.W., and Moffatt, B.A. (1986) Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Clined Genes. J. Mol. Biol. 189, 113-130

Towbin, H., Staehelin, T., and Gordon, J. (1992) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnol. 24, 145-149.

Tsai, K.J., Tsai, Y.C., and James Shen, C.K. (2007) G-CSF rescues the memory impairment of animal models of  Alzheimer's disease . The Journal of Experimental Medicine. 204, 1273-1280

Wolff, A.M., Hansen, O.C., Poulsen, U., Madrid, S., and Stougaard, P. (2001) Optimization of the production of chondrus crispus hexose oxidase in P.pastoris. Protein Expr. Purif. 22, 189-199.

Woo, J.H., Liu, Y.Y., Mathias, A., Stavrou, S., Wang, Z., Thompson, J., and Neville, D.M. (2002) Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris. Protein Expr.Purif. 25, 270-282.

Yamaguchi, T., Yamaguchi, T., Kogi, M., Yamamoto, Y., and Hayakawa, T. (1997) Bioassay of human granulocyte colony-stimulating factor using human promyelocytic HL-60 cells. Biol. Harm. Bull. 20, 943-947.