An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography

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	An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography
	Wang, Jun 1,2; Wang, Yinjue 1; Hu, Tao 1; Li, Xiunan 1; Huang, Yongdong 1; Liu, Yongdong 1; Ma, Guanghui 1; Su, Zhiguo 1
刊名	PROCESS BIOCHEMISTRY
	2012
卷号	47 期号:1 页码:106-112
关键词	PEGylation Staphylokinase Solid phase Immobilized metal-ion affinity chromatography
ISSN号	1359-5113
通讯作者	Liu, YD
英文摘要	Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20 kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10 kDa- and 20 kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5 kDa-SC-mPEG. Bioactivities of 10 kDa- and 20 kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy. (C) 2011 Elsevier Ltd. All rights reserved.
WOS标题词	Science & Technology ; Life Sciences & Biomedicine ; Technology
类目[WOS]	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering, Chemical
研究领域[WOS]	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology ; Engineering
关键词[WOS]	POLYETHYLENE-GLYCOL ; PLASMINOGEN ACTIVATION ; PROTEIN PEGYLATION ; HEMOGLOBIN ; PEPTIDE ; DERIVATIVES ; ATTACHMENT
收录类别	SCI
语种	英语
WOS记录号	WOS:000300133000015
公开日期	2013-10-28
内容类型	期刊论文
版本	出版稿
源URL	[http://ir.ipe.ac.cn/handle/122111/4309]
专题	过程工程研究所_生化工程国家重点实验室
作者单位	1.Chinese Acad Sci, Natl Key Lab Biochem Engn, Inst Proc Engn, Beijing 100190, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100190, Peoples R China
推荐引用方式 GB/T 7714	Wang, Jun,Wang, Yinjue,Hu, Tao,et al. An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography[J]. PROCESS BIOCHEMISTRY,2012,47(1):106-112.
APA	Wang, Jun.,Wang, Yinjue.,Hu, Tao.,Li, Xiunan.,Huang, Yongdong.,...&Su, Zhiguo.(2012).An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography.PROCESS BIOCHEMISTRY,47(1),106-112.
MLA	Wang, Jun,et al."An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography".PROCESS BIOCHEMISTRY 47.1(2012):106-112.