Visualizing and analyzing protein displacement in ion exchange adsorbent

doi:10.3969/j.issn.0438-1157.2013.01.039

CIESC Journal ›› 2013, Vol. 64 ›› Issue (1): 340-345.DOI: 10.3969/j.issn.0438-1157.2013.01.039

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Visualizing and analyzing protein displacement in ion exchange adsorbent

SHI Zhicong, SHI Qinghong, SUN Yan

Department of Biochmeical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Revised:2012-09-06 Online:2013-01-05 Published:2013-01-05

离子交换介质内蛋白置换行为的观测与解析

石智聪, 史清洪, 孙彦

天津大学化工学院生物化工系,天津 300072

通讯作者: 史清洪
作者简介:石智聪(1986—),女,硕士研究生。
基金资助:
国家自然科学基金项目(20976126, 21276189);天津市应用基础研究计划项目(09JCYBJC06800);教育部新世纪优秀人才支持计划项目(NCET-08-0387)。

Abstract

Abstract: The displacement behavior of enhanced green fluorescent protein (eGFP) on Q Sepharose HP was investigated using phytic acid sodium salt hydrate (PAS) as the displacer.The protein displacement in chromatographic column indicated that a stable displacement train of eGFP could be developed at different PAS concentrations, and subsequently a successful separation of eGFP was achieved.Furthermore, eGFP adsorption and displacement on the particle scale were also visualized by confocal laser scanning microscopy (CLSM) and further analyzed to elucidate their mechanism.CLSM images in protein adsorption exhibited that mass transfer of eGFP inside Q Sepharose HP was a complicated phenomenon controlled by both surface and pore diffusion.Moreover, CLSM images in the displacement process on the particle scale indicated that the displacement of eGFP by PAS not only depended on the affinity of the displacer but also was related to the kinetics of PAS binding.Finally, the trajectory of radial fluorescent distribution inside the adsorbent illustrated that eGFP displacement occurred firstly at the outmost layer of the adsorbent and a decrease in displacer concentration led to a drop of fluorescent fading rate.It was consistent with the results in displacement chromatography of eGFP.It was confirmed that CLSM provided an intuitive tool to visualize the intraparticle displacement of protein, and gained insight into the complexity of protein displacement at the particle scale.

Key words: displacement chromatography, confocal laser scanning microscopy, green fluorescent protein, intraparticle mass transfer, phytic acid sodium salt hydrate

摘要： 以肌醇六磷酸钠(PAS)为置换剂研究了绿色荧光蛋白(eGFP)在Q Sepharose HP离子交换色谱(IEC)介质上的置换行为。置换色谱结果表明,不同浓度PAS下eGFP均可形成稳定的置换列并成功分离。利用共聚焦激光扫描显微镜(CLSM)技术对eGFP在离子交换色谱介质内吸附动力学的观测发现,eGFP在IEC介质内的吸附是由表面扩散和孔扩散共同作用的复杂行为。置换过程中不同时刻的CLSM图像显示,eGFP置换不仅取决于置换剂的亲和力,也与置换剂与介质的结合过程相关;介质内荧光强度的分析表明,eGFP置换首先发生在介质的外层,PAS浓度的降低导致置换速率下降。这与置换色谱的结果相吻合。CLSM不仅为蛋白置换动力学的研究提供了一种直观观测技术,CLSM结果也确认了蛋白质置换过程的复杂性。

关键词: 置换色谱, 共聚焦激光扫描显微镜, 绿色荧光蛋白, 介质内传质, 肌醇六磷酸钠

CLC Number:

TQ936.2

SHI Zhicong, SHI Qinghong, SUN Yan. Visualizing and analyzing protein displacement in ion exchange adsorbent[J]. CIESC Journal, 2013, 64(1): 340-345.

石智聪, 史清洪, 孙彦. 离子交换介质内蛋白置换行为的观测与解析[J]. 化工学报, 2013, 64(1): 340-345.

References 17

[1]	Ljunglf A, Hjorth R.Confocal microscopy as a tool for studying protein adsorption to chromatographic matrices[J].J.Chromatogr A, 1996,743(1):75-83
[2]	Linden T, Ljunglof A, Hagel L, Kula M R, Thommes J.Visualizing patterns of protein uptake to porous media using confocal scanning laser microscopy[J].Sep. Sci. Technol., 2002,37(1):1-32
[3]	Dziennik S R, Belcher E B, Barker G A, DeBergalis M J, Fernandez S E, Lenhoff A M.Nondiffusive mechanisms enhance protein uptake rates in ion exchange particles[J].Proc.Nat.Acad.Sci., 2003,100(2):420-425
[4]	Yang K, Bai S, Sun Y.Protein adsorption dynamics in cation-exchange chromatography quantitatively studied by confocal laser scanning microscopy[J].Chem.Eng.Sci., 2008,63(16):4045-4054
[5]	Zhao G F, Zhang L, Bai S, Sun Y.Analysis of hydrophobic charge induction displacement chromatography by visualization with confocal laser scanning microscopy[J].Sep.Purif.Technol., 2011,82:138-147
[6]	Teske C A, von Lieres E, Schroder M, Ladiwala A, Cramer S M, Hubbuch J J.Competitive adsorption of labeled and native protein in confocal laser scanning microscopy[J].Biotechnol.Bioeng., 2006,95(1):58-66
[7]	Harinarayan C, Mueller J, Ljunglof A, Fahrner R, van Alstine J, van Reis R.An exclusion mechanism in ion exchange chromatography[J].Biotechnol.Bioeng., 2006,95(5):775-787
[8]	Mazza C B, Rege K, Breneman C M, Sukumar N, Dordick J S, Cramer S M.High-throughput screening and quantitative structure-efficacy relationship models of potential displacer molecules for ion-exchange systems[J].Biotechnol.Bioeng., 2002,80(1):60-72
[9]	Shi Qinghong(史清洪), Yang Jie(杨婕), Su Liang(苏亮), Sun Yan(孙彦).Synthesis and characterization of novel amino acid-based dendritic displacers[J].J. Chem. Ind.Eng.(China)(化工学报), 2007,58(5):1271-1276
[10]	Zhao G, Bai S, Sun Y.Development of a displacer-immobilized ligand docking scheme for displacer screening for protein displacement chromatography[J].Biochem. Eng. J., 2011,55(1):32-42
[11]	Morrison C J, Godawat R, McCallum S A, Garde S, Cramer S M.Mechanistic studies of displacer-protein binding in chemically selective displacement systems using NMR and MD simulations[J].Biotechnol.Bioeng., 2009,102(5):1428-1437
[12]	Golshan-Shirazi S, Lin B, Guiochon G.Influence of the mass transfer kinetics on the separation of a binary mixture in displacement liquid chromatography[J].Anal.Chem., 1989,61(17):1960-1970
[13]	Natarajan V, Cramer S M.Modeling shock layers in ion-exchange displacement chromatography[J].AIChE J., 1999,45(1):27-37
[14]	Shi Q H, Shi Z C, Sun Y.Dynamic behavior of binary component ion-exchange displacement chromatography of proteins visualized by confocal laser scanning microscopy[J].J.Chromatogr A, 2012,1257:48-57
[15]	Dong X Y, Chen L J, Sun Y.Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography[J].J.Chromatogr A, 2009,1216(27):5207-5213
[16]	Evans S I, Freed A, Cramer S M.Displacer concentration effects in displacement chromatography implications for trace solute detection[J].J.Chromatogr A, 2009,1216(1):79-85
[17]	Yang K, Shi Q H, Sun Y.Modeling and simulation of protein uptake in cation exchanger visualized by confocal laser scanning microscopy[J].J.Chromatogr A, 2006,1136(1):19-28

Visualizing and analyzing protein displacement in ion exchange adsorbent

离子交换介质内蛋白置换行为的观测与解析

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[1]	ZHANG Chong, XING Xinhui. Fluorescent Proteins as a Visible Molecular Signal for Rapid Quantification of Bioprocesses:Potential and Challenges [J]. , 2010, 18(5): 863-869.
[2]	CHEN ,Yin, XING ,Xinhui, YE ,Fengchun, KUANG ,Ying. Soluble expression and rapid quantification of GFP-hepA fusion protein in recombinant Escherichia coli [J]. , 2007, 15(1): 122-126.
[3]	ZHOU Xiaopeng, SHI Qinghong, XING Xinhui, SUN Yan. Rapid Purification of Enhanced Green Fluorescent Protein from Escherichia coli [J]. , 2006, 14(2): 229-234.