Affinity Purification of Tumor Necrosis Factor-alpha Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

被引：9

作者：

Abdolalizadeh, Jalal ^{[1
,2
]}

Zolbanin, Jafar Majidi ^{[3
,4
]}

Nouri, Mohammad ^{[5
]}

Baradaran, Behzad ^{[4
]}

Movassaghpour, AliAkbar ^{[6
]}

Farajnia, Safar ^{[7
]}

Omidi, Yadollah ^{[1
,8
]}

机构：

[1] Tabriz Univ Med Sci, Res Ctr Pharmaceut Nanotechnol, Tabriz, Iran

[2] Tabriz Univ Med Sci, Student Res Comm, Tabriz, Iran

[3] Tabriz Univ Med Sci, Drug Appl Res Ctr, Tabriz, Iran

[4] Tabriz Univ Med Sci, Immunol Res Ctr, Tabriz, Iran

[5] Tabriz Univ Med Sci, Med Fac, Biochem Dept, Tabriz, Iran

[6] Tabriz Univ Med Sci, Hematol & Oncol Res Ctr, Tabriz, Iran

[7] Tabriz Univ Med Sci, Biotechnol Res Ctr, Tabriz, Iran

[8] Univ Penn, Ovarian Canc Res Ctr, Translat Res Ctr, Philadelphia, PA 19104 USA

来源：

ADVANCED PHARMACEUTICAL BULLETIN | 2013年 / 3卷 / 01期

关键词：

TNF-alpha expression; Affinity Purification; Monoclonal antibody; LPS;

D O I：

10.5681/apb.2013.004

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

Recombinant tumor necrosis factor-alpha (TNF-alpha) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-alpha scFv fragments for purification of TNF-alpha produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-a. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-alpha expression. The anti-TNF-alpha scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-alpha and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-alpha with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-alpha protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-alpha protein can be applied for various in vitro and in vivo applications.

引用

页码：19 / 23

页数：5

共 25 条

[1]

Abdolalizadeh Jalal, 2012, Human Antibodies, V21, P41, DOI 10.3233/HAB-2012-0260

[2] Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins [J].