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Prevention of aggregate formation through mechanism analysis in refolding of recombinant pertactin from Escherichia coli
Li, Zenglan1,2; Zhang, Chun1; Zhang, Yan1; Liu, Yongdong1; Li, Xiunan1; Ma, Guanghui1; Luo, Jim1; Su, Zhiguo1,3
2017-11-01
Source PublicationPROCESS BIOCHEMISTRY
ISSN1359-5113
Volume62Issue:00Pages:80-90
Abstract

Production of natural pertactin for pharmaceutical use is limited by its low abundance. In this study, recombinant pertactin was highly expressed in the form of inclusion bodies in E. coll. However, up to 75% of the soluble turned out as aggregates when refolding by pulse-fed batch dilution. The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein, resulting in aggregation between proteins with different folding states. The key factor for prevention of aggregate formation was to improve the synchronization of refolding. For this purpose, flash-batch dilution was conducted at a scale of 5 L and achieved a monomeric refolding yield of above 70%. Aggregates formed were efficiently removed along with impurities by one-step chromatography of Ni-resin. The purity of monomeric pertactin was > 98%. An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%. The purified protein was characterized by MALDI-TOF, circular dichroism, fluorescence, HPLC and DSC, and showed similar physiochemical properties compared to its natural counterpart. Animal study showed similar immunological responses and antibodies elicited demonstrated a comparable reactivity.

KeywordRecombinant Pertactin Refolding Aggregates Dilution
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Technology
DOI10.1016/j.procbio.2017.07.001
Indexed BySCI
Language英语
WOS KeywordAcellular Pertussis-vaccine ; Factor P.69 Pertactin ; Bordetella-pertussis ; Immunological-properties ; Protective Properties ; Protein ; Expression ; Antibodies
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering, Chemical
Funding OrganizationNational Basic Research Program(2013CB733604) ; Natural Science Foundation of China(21336010)
WOS IDWOS:000416394500010
Citation statistics
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/23512
Collection研究所(批量导入)
Corresponding AuthorLuo, Jim; Su, Zhiguo
Affiliation1.Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Jiangsu Natl Synerget Innovat Ctr Adv Mat, Nanjing 211800, Jiangsu, Peoples R China
Recommended Citation
GB/T 7714
Li, Zenglan,Zhang, Chun,Zhang, Yan,et al. Prevention of aggregate formation through mechanism analysis in refolding of recombinant pertactin from Escherichia coli[J]. PROCESS BIOCHEMISTRY,2017,62(00):80-90.
APA Li, Zenglan.,Zhang, Chun.,Zhang, Yan.,Liu, Yongdong.,Li, Xiunan.,...&Su, Zhiguo.(2017).Prevention of aggregate formation through mechanism analysis in refolding of recombinant pertactin from Escherichia coli.PROCESS BIOCHEMISTRY,62(00),80-90.
MLA Li, Zenglan,et al."Prevention of aggregate formation through mechanism analysis in refolding of recombinant pertactin from Escherichia coli".PROCESS BIOCHEMISTRY 62.00(2017):80-90.
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