Improved production of recombinant human Fas ligand extracellular domain in Pichia pastoris: yield enhancement using disposable culture-bag and its application to site-specific chemical modifications

BMC Biotechnology

Michiro Muraki¹ 

[1] Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan

关键词: Site-specific chemical modification;    Yield enhancement;    Pichia pastoris;    Production;    Extracellular domain;    Human Fas ligand;   

Others  :  834891
DOI  :  10.1186/1472-6750-14-19

 received in 2013-06-25, accepted in 2014-03-03,  发布年份 2014

【 摘 要 】

Background

A useful heterologous production system is required to obtain sufficient amounts of recombinant therapeutic proteins, which are often necessary for chemical characterization and engineering studies on the development of molecules with improved properties. Human Fas ligand extracellular domain (hFasLECD) is an agonistic death ligand protein that has potential applications for medical purposes. Site-specific chemical modifications can provide a powerful means for the development of engineered proteins with beneficial functions. This study aimed to enhance the yield of hFasLECD using a Pichia pastoris secretory expression system suitable for efficient production on a small laboratory scale, and further to provide procedures for its site-specific chemical modification without impairing the biological functions based on the developed production system.

Results

A convenient cultivation system using a disposable plastic bag provided a three-fold increase in purification yield of tag-free hFasLECD as compared with the conventional system using a baffled glass flask. The system was further applied to the production of a mutant, which contains an additional reactive cysteine residue in the N-terminal tag-sequence region. Site-specific conjugations and cross-linking without impairing biological functions were achieved by reaction of the mutant hFasLECD with single maleimide group containing compounds and a linear polyethylene glycol derivative containing two maleimide groups at either end, respectively. All purified tag-free and chemically modified hFasLECDs showed an evident receptor binding activity in co-immunoprecipitation experiments mediated by wild-type and N-glycosylation site deficient mutant human Fas receptor extracellular domain derivatives. An N-Ethylmaleimide conjugated hFasLECD derivative demonstrated a significant cytotoxic activity against human HT-29 colorectal cancer cells.

Conclusions

A new, efficient cultivation system for enhanced secretory production of hFasLECD using P. pastoris and an effective strategy for site-specific chemical modifications of hFasLECD were devised. The results obtained constitute the basis for biomedical applications including developments of novel therapeutic proteins and diagnostic tools targeted to related diseases and their biomarkers.

【 授权许可】

   
2014 Muraki; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Nagata S: Apoptosis by death factor. Cell 1997, 88:355-365.
• [2]Ashkenazi A: Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov 2008, 7:1001-1012.
• [3]Vinay DS, Kwon BS: The tumor necrosis factor/TNF receptor superfamily: therapeutic targets in autoimmune diseases. Clin Exp Immunol 2011, 164:145-157.
• [4]Takahashi T, Tanaka M, Inazawa J, Abe T, Suda T, Nagata S: Human Fas ligand: gene structure, chromosomal location and species specificity. Int Immunol 1994, 6:1567-1574.
• [5]Ferrer-Miralles N, Domingo-Espín J, Corchero JL, Vázquez E, Villaverde A: Microbial factories for recombinant pharmaceuticals. Microb Cell Fact 2009, 8:17. BioMed Central Full Text
• [6]Muraki M: Heterologous production of death ligands’ and death receptors’ extracellular domains: structural features and efficient systems. Protein Pept Lett 2012, 19:867-879.
• [7]Luo Z, Xu Z, Zhuo S, Jing K, Lu Y: Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum. Biochem Engineer J 2012, 62:86-91.
• [8]Tanaka M, Suda T, Yatomi T, Nakamura N, Nagata S: Lethal effect of recombinant human Fas ligand in mice pretreated with Propionibacterium acnes. J Immunol 1997, 158:2303-2309.
• [9]Muraki M: Secretory expression of synthetic human Fas ligand extracellular domain gene in Pichia pastoris: influences of tag addition and N-glycosylation site deletion, and development of a purification method. Protein Expr Purif 2006, 50:137-146.
• [10]Muraki M: Improved secretion of human Fas ligand extracellular domain by N-terminal part truncation in Pichia pastoris and preparation of the N-linked carbohydrate chain trimmed derivative. Protein Expr Purif 2008, 60:205-213.
• [11]Harris JM, Chess RB: Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2003, 2:214-221.
• [12]Muraki M, Jigami Y, Harata K: Alteration of the substrate specificity of human lysozyme by site-specific intermolecular cross-linking. FEBS Lett 1994, 355:271-274.
• [13]Muraki M, Harata K, Sugita N, Sato KI: Origin of carbohydrate recognition specificity of human lysozyme revealed by affinity labeling. Biochemistry 1996, 35:13562-13567.
• [14]Muraki M, Honda S: Efficient production of human Fas receptor extracellular domain-human IgG1 heavy chain Fc domain fusion protein using baculovirus/silkworm expression system. Protein Expr Purif 2010, 73:209-216.
• [15]Muraki M, Honda S: Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus – silkworm expression system. Protein Expr Purif 2011, 80:102-109.
• [16]Ebert EC, Groh V: Dissection of spontaneous cytotoxicity by human intestinal intraepitherial lymphocytes: MIC on colon cancer triggers NKG2D-mediated lysis through Fas ligand. Immunology 2008, 124:33-41.
• [17]Shukla AA, Gottschalk U: Single-use disposable technologies for biopharmaceutical manufacturing. Trends Biotech 2013, 31:147-154.
• [18]Higgins DR, Cregg JM: Pichia Protocols. Totowa NJ: Humana Press; 1998. [[Methods in Molecular Biology Vol. 103]]
• [19]Schneider P, Bodmer JL, Holler N, Mattmann C, Scuderi P, Terskikh A, Peitsch MC, Tschopp J: Characterization of Fas (Apo-1, CD95)-Fas ligand interaction. J Biol Chem 1997, 272:18827-18833.
• [20]Orlinick JR, Elkon KB, Chao MV: Separate domains of the human Fas ligand dictate self-association and receptor binding. J Biol Chem 1997, 272:32221-32229.
• [21]Bajorath J: Identification of the ligand binding site in Fas (CD95) and analysis of Fas-ligand interactions. Proteins 1999, 35:475-482.
• [22]Shatnyeva OM, Kubarenko AV, Weber CEM, Pappa A, Schwartz-Albiez R, Weber ANR, Krammer PH, Lavrik IN: Modulation of the CD95-induced apoptosis: the role of CD95 N-glycosylation. PLoS ONE 2011, 6:e19927.
• [23]Nett JH, Gomathinayagam S, Hamilton SR, Gong B, Davidson RC, Du M, Hopkins D, Mitchell T, Mallem MR, Nylen A, Shaikh SS, Sharkley N, Barnard GC, Copeland V, Liu L, Evers R, Li Y, Gray PM, Lingham RB, Visco D, Forrest G, DeMartino J, Linden T, Potgieter TI, Wildt S, Stadheim TA, d’Anjou M, Li H, Sethuraman N: Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris. J Biotechnol 2012, 157:198-206.
• [24]Liu W, Ramagopal UA, Zhan C, Bonanno JB, Bhosle RC, Nathenson SG, Almo SC, Atoms-to-Animals: The immune Function Network (IFN), New York Structural Genomics Research Consortium (NYSGRC): Crystal Structure of FASL and DcR3 complex. [ http://pdbj.org/mine/summary/4msv webcite]
• [25]Hermanson GT: Bioconjugate Techniques. 2nd edition. London: Academic Press; 2008.
• [26]Chalker JM, Bernardes GJL, Davis BG: A “Tag-and-Modify” approach to site-selective protein modification. Acc Chem Res 2011, 44:730-741.
• [27]Backer MV, Levashova Z, Levenson R, Blankenberg FG, Backer JM: Cysteine-containing fusion tag for site-specific conjugation of therapeutic and imaging agents to targeting proteins. Methods Mol Biol 2008, 494:275-294.
• [28]Cong Y, Pawlisz E, Bryant P, Balan S, Laurine E, Tommasi R, Singh R, Dubey S, Peciak K, Bird M, Sivasanker A, Swierkosz J, Muroni M, Heidelberger S, Farys M, Khayrzad F, Edwards J, Badescu G, Hodgson I, Heise C, Somavarapu S, Liddell J, Powell K, Zloh M, Choi JW, Godwin A, Brocchini S: Site-specific PEGylation at histidine tags. Bioconj Chem 2012, 23:248-263.
• [29]Holler N, Tardivel A, Kovacsovics-Bankowski M, Hertig S, Gaide O, Martinon F, Tinel A, Deperthes D, Calderara S, Schulthess T, Engel J, Schneider P, Tschopp J: Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Mol Cell Biol 2003, 23:1428-1440.
• [30]Bremer E, Cate BT, Samplonius DF, Mueller N, Wajant H, Stel AJ, Chamuleau M, van de Loosdrecht AA, Stieglmaier J, Fey GH, Helfrich W: Superior activity of fusion protein scFvRit:sFasL over cotreatment with rituximab and Fas agonists. Cancer Res 2008, 68:597-604.
• [31]Chan DV, Sharma R, Ju C-Y A, Roffler SR, Ju S-T: A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer. J Biomed Sci 2013, 20:16. BioMed Central Full Text
• [32]Connolly K, Cho YH, Duan R, Fikes J, Gregorio T, Lafleuer DW, Okoye Z, Salcedo TW, Santiago G, Ullrich S, Wei P, Windle K, Wong E, Yao XT, Zhang YQ, Zheng G, Moore PA: In vivo inhibition of Fas ligand-mediated killing by TR6, a Fas ligand decoy receptor. J Pharmcol Exp Therapeutics 2001, 298:25-33.
• [33]Linkermann A, Qian J, Lettau M, Kabelitz D, Janssen O: Considering Fas ligand as a target for therapy. Expert Opin Ther Targets 2005, 9:119-134.
• [34]Lin WW, Hsieh SL: Decoy receptor 3: a pleiotropic immunomodulator and biomarker for inflammatory diseases, autoimmune diseases and cancer. Biochem Pharmacol 2011, 81:838-847.
• [35]Yamana K, Bilim V, Hara N, Kasahara T, Itoi T, Maruyama R, Nishiyama T, Takahashi K, Tomita Y: Prognostic impact of FAS/CD95/APO-1 in urotherial cancers: decreased expression of Fas is associated with disease progression. Brit J Cancer 2005, 93:544-551.
• [36]Kim Y, Ho SO, Gassman NR, Korlann Y, Landorf EV, Collart FR, Weiss S: Efficient site-specific labeling of proteins via cysteines. Bioconj Chem 2008, 19:786-791.
• [37]Inouye S, Sato JI: Recombinant aequorin with a reactive cysteine residue for conjugation with maleimide-activated antibody. Anal Biochem 2008, 378:105-107.
• [38]Boroumand-Noughabi S, Sima HR, Ghaffarzadehgan K, Jafarzadeh M, Raziee HR, Hosseinnezhad H, Moaven O, Rajabi-Mashhabi MT, Azarian AA, Mashhadinejad M, Tavakkol-Afshari J: Soluble Fas might serve as a diagnostic tool for gastric adenocarcinoma. BMC Cancer 2010, 10:275. BioMed Central Full Text
• [39]Furuya Y, Nagakawa O, Fuse H: Prognostic significance of serum soluble Fas level and its change during regression and progression of advanced prostate cancer. Endocrine J 2003, 50:629-633.
• [40]Takahama Y, Yamada Y, Emoto K, Fujimoto H, Takayama T, Ueno M, Uchida H, Hirao S, Mizuno T, Nakajima Y: The prognostic significance of overexpression of the decoy receptor for Fas ligand (DcR3) in patients with gastric carcinomas. Gastric Cancer 2002, 5:61-68.
• [41]Reimer T, Koczan D, Müller H, Friese K, Thiesen HJ, Gerber B: Tumor Fas ligand: fas ratio greater than 1 is an independent marker of relative resistance to tamoxifen therapy in hormone receptor positive breast cancer. Breast Cancer Res 2002, 4:R9. BioMed Central Full Text
• [42]Tamakoshi A, Nakachi K, Ito Y, Lin Y, Yagyu K, Kikuchi S, Watanabe Y, Inaba Y, Tajima K: Soluble Fas level and cancer mortality: findings from a nested case-control study within a large-scale prospective study. Int J Cancer 2008, 123:1913-1916.