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Selection of downstream steps by analysis of protein surface property: A case study for recombinant human lactoferrin purification from milk of transgenic cow
Zhang, Yan; Luo, Jian; Zhang, Songping; Bai, Qian; Li, Zenglan; Li, Qiang; Liu, Yongdong; Zhang, Guifeng; Ma, Guanghui; Su, Zhiguo
2015-09-01
Source PublicationPROCESS BIOCHEMISTRY
ISSN1359-5113
Volume50Issue:9Pages:1441-1448
Abstract

Protein purification has long been regarded as an art rather than a science. Trial and error is the widely used practice for a protein to be purified from the crude extract. In this study, we carried out a rational approach by analysis of the protein surface properties before setting out to do experiments. The target protein was recombinant human lactoferrin (rHLF), to be purified from milk of transgenic cow. We need to overcome two major problems. One is its possible co-precipitation with casein during initial separation; the other is the difficulty in separating its homologous counterpart, bovine lactoferrin (BLF). By calculation of the surface hydrophobicity, an initial separation step was decided by calcium precipitation, which removed casein but left rHLF in the supernatant. Then the average surface hydrophobicity and electric potential of rHLF were compared with those of BLF. There was a more significant difference in the electric potentials than the average surface hydrophobicity (ASH) between the homologous pairs. Therefore, the purification step should be favored by ion exchange chromatography (IEC) instead of hydrophobic interaction chromatography (HIC). Experiments were performed to verify the prediction. After removing casein, one step cationic ion exchange chromatography realized complete separation of rHLF from BLF with rHLF recovery up to 83%, while the resolution of HIC process was very limited due to the small difference in ASH between them. The laboratory process was then successfully scaled up to pilot-plant scale of 5001 milk of transgenic cow per batch. Average rHLF recovery of 79%and purity of 98.9% were attained for five batches. The purified rHLF displayed bioactivities as good as the natural human-resource lactoferrin standard. (C) 2015 Elsevier Ltd. All rights reserved.

KeywordSeparation Homologous Proteins Lactoferrin Milk From Transgenic Cow Pilot Scale
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Technology
DOI10.1016/j.procbio.2015.05.025
Indexed BySCI
Language英语
WOS KeywordHYDROPHOBIC INTERACTION CHROMATOGRAPHY ; AMINO-ACID DISTRIBUTION ; LARGE-SCALE PRODUCTION ; VIRUS-LIKE PARTICLES ; NERVE GROWTH-FACTOR ; EXCHANGE CHROMATOGRAPHY ; PICHIA-PASTORIS ; KAPPA-CASEIN ; EXPRESSION ; SEPARATION
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering, Chemical
WOS IDWOS:000359166400016
Citation statistics
Cited Times:5[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/19423
Collection生化工程国家重点实验室
AffiliationChinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing 100190, Peoples R China
Recommended Citation
GB/T 7714
Zhang, Yan,Luo, Jian,Zhang, Songping,et al. Selection of downstream steps by analysis of protein surface property: A case study for recombinant human lactoferrin purification from milk of transgenic cow[J]. PROCESS BIOCHEMISTRY,2015,50(9):1441-1448.
APA Zhang, Yan.,Luo, Jian.,Zhang, Songping.,Bai, Qian.,Li, Zenglan.,...&Su, Zhiguo.(2015).Selection of downstream steps by analysis of protein surface property: A case study for recombinant human lactoferrin purification from milk of transgenic cow.PROCESS BIOCHEMISTRY,50(9),1441-1448.
MLA Zhang, Yan,et al."Selection of downstream steps by analysis of protein surface property: A case study for recombinant human lactoferrin purification from milk of transgenic cow".PROCESS BIOCHEMISTRY 50.9(2015):1441-1448.
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