Expression and Purification of Proliferating Cell Nuclear Antigen in <i>Spodoptera frugiperda</i> Cells

doi:10.13523/j.cb.20180703

China Biotechnology

2018, Vol. 38

Issue (7): 14-20 DOI: 10.13523/j.cb.20180703

Expression and Purification of Proliferating Cell Nuclear Antigen in Spodoptera frugiperda Cells

Jun-jun CHEN¹,Ying LOU¹,Yuan-xing ZHANG^1,^2,³,Qin LIU^1,^2,³,Xiao-hong LIU^1,^2,^**(

)

1 East China University of Science and Technology, Shanghai 200237, China
2 Shanghai Engineering Research Centers of Maricultured Animal Vaccines, Shanghai 200237, China
3 Shanghai Collaborative Innovation Center for Biomanufacturing, Shanghai 200237, China

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Abstract

Objetive: The object is to produce recombinant human proliferating cell nuclear antigen (PCNA) protein using insect cell expression system and purify and identify its antibody binding characterization. Methods: Human PCNA gene was amplified from HeLa cells and cloned into the baculovirus vector AcMNPV. Using insect cells, the recombinant baculovirus containing the PCNA gene was obtained. The virus infected the cells to express the protein, which was reached high purity by combining the nickel column affinity chromatography and the ion exchange chromatography. ELISA method was set to identify its binding activity. Results: Full length recombinant human PCNA (rPCNA) was produced in a baculovirus expression system. The optimal multiplicity of infection (MOI) value and infected time were 0.05h and 144h respectively. The produced protein samples were subsequently purified by a two-step procedure, including Ni-NTA affinity chromatography and ion exchange chromatography. The yield of rPCNA was up to 110mg/L cell culture, with a purity > 95% by SDS-PAGE. Indirect ELISA results showed that antibody binding activity of rPCNA was much higher than that of PCNA expressed by E. coli and rPCNA took a sensitivity and specificity of 93.3% and 85.0%, respectively. Conclusions: An expression and purification procedure for rPCNA and the produced rPCNA presented high antibody binding characterization were established which would have great potential applications on diagnosis of PCNA-associated diseases in vitro.

Key words： Proliferating cell nuclear antigen Baculovirus Insect cells Protein purification Antibody binding characterization

Received: 26 February 2018 Published: 13 August 2018

ZTFLH:

Q819

Corresponding Authors: Xiao-hong LIU E-mail: liuxiaohong@ecust.edu.cn

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	Jun-jun CHEN
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Jun-jun CHEN,Ying LOU,Yuan-xing ZHANG,Qin LIU,Xiao-hong LIU. Expression and Purification of Proliferating Cell Nuclear Antigen in Spodoptera frugiperda Cells. China Biotechnology, 2018, 38(7): 14-20.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180703 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I7/14

Fig.1 Generation of recombinant baculovirus for expressing PCNA by Bac-to-Bac system (a) Sequence alignment of the sub-cloned gene of PCNA with the theoretical sequence from GenBank 1: Theoretical sequence;2: Tested sequence (b) The expression cassette was applied for producing rPCNA protein (c) Sf9 insect cells were infected with buffer (mock, C1) or P₃ baculovirus (C2) (d) Three days post infection, protein PCNA was tested by western blot with anti-His antibody in cell-only control and infected cells

Fig. 2 Effect of MOI and infection time on expression of rPCNA Sf9 cells in 6-well plates were infected with P₃ baculovirus at different MOI values (0,0.05, 0.2, 1, 4, 10) for different periods (24h, 48h, 72h, 96h, 120h, 144h). Cell pellets were tested by Western blot with anti-His antibody

Fig.3 Small-scale expression and purification of rPCNA by Ni-NTA chromatograph (a) Proteins hanging on the column were eluted by imidazole gradient (50mmol/L, 100mmol/L, 200mmol/L, 500mmol/L) and tested by silver staining assay (b)Aliquots eluted from 200mmol/L and 500mmol/L imidazole were concentrated and analyzed by SDS-PAGE gel stained with coomassie brilliant blue staining and Western blot with anti-PCNA antibody(c) Lane M: Standard protein marker; Lane 1, 2: The extracellular medium and the cell pellets of cell-only, respectively; Lane 3: The cell lysate of infected cells; Lane 4: Flow-through; Lane 5: Effluent of 20mmol/L imidazole; Lane P₁: Standard protein of PCNA (from Diarect Company); Lane P₂: rPCNA purified by Ni-NTA column

Fig. 4 Large-scale expression and purification of rPCNA (a),(b)Cell lysate was purified by Ni-NTA chromatograph (c)-(e)The clean fractions of Ni-NTA chromatograph were further purified by ion exchange column (Q sepharose) All rPCNA was eluted by 400mmol/L sodium chloride(c) and transferd to preservation solution (P₂);additionally detected by coomassie blue staining (d) and Western blot (e) Lane M: Standard protein marker; Lane 1: Cell pellets of cell-only; Lane P₁: Standard protein of PCNA; P₂: Purified protein of rPCNA

Table 1 ELISA detecting the binding specificity to antibodies of rPCNA

Fig.5 Binding specificity test of recombinant PCNA by indirect ELISA P: Positive serum of SLE; N: Negative serum of SLE; PCNA1: PCNA product expressed by E. coli (from Diarect Company); rPCNA: Recombinant PCNA


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