Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology  2011, Vol. 31 Issue (8): 66-72    DOI:
    
Purification of Recombinant Human Lactoferrin Expressed in A Cattle Mammary Bioreactor
LI You1,2, ZHANG Yan2, SU Hai-jia1, LUO Jian2, BAI Qian1, MA Guang-hui2, SU Zhi-guo2
1. College of Life Science and Technology,Beijing University of Chemical Technology,Beijing 100029,China;
2. National Key Laboratory of Biochemistry Engineering,Chinese Academy of Sciences,Beijing 100190,China
Download: HTML   PDF(920KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

Recombinant human α-lactalbumin(rHLA)could be expressed in mammary gland bioreactor effectively. However,it is very difficult to purify the target protein. Primary proteins’ properties,such as surface hydrophobicity and electric potential,were compared through molecular simulation and calculation,based on which a fast purification process with high resolution was designed. Immunoglobulin G,which would disturb chromatographic purification process,was firstly removed using ammonium sulfate precipitation with orthogonal optimization. Then the subsequent hydrophobic interaction chromatography(HIC)became more stable and rHLA with 95% purity was obtained through separating successfully from its homologous courterpart,i.e.,bovine α-lactalbumin in HIC. The overall recovery of rHLA was up to 48.6%. Activity detection and circular dichroism spectroscopy(CD)confirmed the regulatory activity for β-1,4-galactosyltransferase and native structure of purified rHLA.



Key wordsMammary gland bioreactor      α-lactalbumin      Hydrophobic interaction chromatography      Homologous proteins     
Received: 28 April 2011      Published: 25 August 2011
ZTFLH:  Q819  
Cite this article:

LI You, ZHANG Yan, SU Hai-jia, LUO Jian, BAI Qian, MA Guang-hui, SU Zhi-guo. Purification of Recombinant Human Lactoferrin Expressed in A Cattle Mammary Bioreactor. China Biotechnology, 2011, 31(8): 66-72.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2011/V31/I8/66


[1] Zhang Y Q. Application and progress in research of animal mammary gland bioreactor. Chin J Biol,2009,22(3):309-312.

[2] Wang J,Yang P,Tang B,et al. Expression and characterization of bioactive recombinant human α-lactalbumin in the milk of transgenic cloned cows. J Dairy Sci,2008,91:4466-4476.

[3] Viaene A,Volckaert G,Joniau M,et al. Efficient expression of bovine α-lactalbumin in Saccharomyces cerevisiae. Eur J Biochem,1991,202(2):471-477.

[4] Saito A,Usui M,Song Y,et al. Secretion of glycosylated α-lactalbumin in yeast Pichia pastoris. J Biochem(Tokyo),2002,132(1):77-82.

[5] Mueller-Dieckmann C,Panjikar S,Schmidt A,et al. On the routine use of soft X-rays in macromolecular crystallography part IV efficient determination of anomalous substructures in biomacromolecules using longer X-ray wavelengths. Acta Crystallogr,Sect D,2007,63:366-380.

[6] Chrysina E D,Brew K,Acharya K R. Crystal structures of apo-and holo-bovine α-lactalbumin at 2,2- resolution reveal an effect of calcium on inter-lobe interactions. J Biol Chem,2000,275:37021-37029.

[7] Chandra N,Brew K,Acharya K R,et al. Structural evidence for the presence of a secondary calcium binding site in human α-lactalbumin. Biochemistry,1998,37:4767-4772.

[8] Eyestone W H. Production and breeding of transgenic cattle using in vitro embryo production technology. Theriogenology,1999,51(2):509-517.

[9] Zhang Y,Luo J,Bi J X,et al. Efficient separation of homologous α-lactalbumin from transgenic bovine milk using optimized hydrophobic interaction chromatography. J Chromatogr A,2010,1217(23):3668-3673.

[10] Skinner M M,Puvathingal J M,Walter R L,et al. Crystal structure of protein isoaspartyl methyltransferase:a catalyst for protein repair. Structure,2000,8:1189-1201.

[11] Salgado J C,Rapaport I,Asenjo J A. Predicting the behaviour of proteins in hydrophobic interaction chromatography 2 using a statistical description of their surface amino acid distribution. J Chromatogr A,2006,1107:110-119.

[12] Rao J K,Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta,1986,869:197-214.

[13] Berggren K,Wolf A,Asenjo J A,et al. The surface exposed amino acid residues of monomeric proteins determine the partitioning in aqueous two-phase systems. Biochim Biophys Acta,2002,1596:253-268.

[14] Frishman D,Argos P. Knowledge-based secondary structure assignment. Proteins,1995,23:566-579.

[15] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature,1970,227:680-685.

[16] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem,1976,72:248-254.

[17] Vijayalakshmi L,Krishna R,Sankaranarayanan R,et al. An asymmetric dimer of β-lactoglobulin in a low humidity crystal form-structural changes that accompany partial dehydration and protein action. Proteins,2007,71:241-249.

[18] Harris L J,Larson S B,Hasel K W,et al. Refined structure of an intact IgG2a monoclonal antibody. Biochemistry,1997,36:1581-1597.

[19] Kuwajima K. The molten globule state of α-lactalbumin. Faseb J,1996,10(1):102-109.

[1] LIANG Zhen-xin,LIU Fang,ZHANG wei,LIU Qing-you,LI Li. The Preparation and Validation of p185 erb B2 Human-mouse Chimeric Antibody ChAb26 Transgenic Mice Mammary Gl and Bioreactor[J]. China Biotechnology, 2019, 39(8): 40-51.
[2] LIANG Zhen-xin, YIN Fu-qiang, LIU Qing-you, LI Li. Progress and Technology in Mammary Gland Bioreactor of Transgenic Animals[J]. China Biotechnology, 2015, 35(2): 92-98.
[3] ZHANG Si-min, GAO Yue, FANG Yu-dan, ZHANG Jin-mai, ZHANG Jin-zhi. Construction of Mammary Gland-specific and Effective Expression Vector for Mammary Gland Bioreactor[J]. China Biotechnology, 2014, 34(7): 49-55.
[4] YAN Ya-bin, CAI Qin, CAI Lin-lin, GONG Xiu-li, ZHU Yi-wen, GUAN Yi-hua, HUANG Ying. Comparison of the Impact on the Transgene Expression Either by Exogenic Bovine Prolactin, or with Growth Hormone[J]. China Biotechnology, 2013, 33(8): 91-97.