- Email: [email protected]
Expression of recombinant human tumor necrosis factor-α in a baculovirus expression system
Bioresource Technology 70 (1999) 299±301
Short communication
Expression of recombinant human tumor necrosis factor-a in a baculovirus expression system Xue-Wu Zhang*, Tao Sun, Xiang-Yue Zeng, Xin Liu, De-Xiang Gu Food Engineering Research Center of State Education Commission, Zhongshan University, Guangzhou 510275, People's Republic of China Received 17 September 1998; received in revised form 2 February 1999; accepted 10 February 1999
Abstract A cDNA fragment coding human tumor necrosis factor-alpha (TNF-a) was inserted into the vector pSXIVVI X3 with the control of Syn XIV promoter. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). Cells infected with recombinant virus synthesized TNF-a protein at a level of about 38% of total cellular protein. TNF-a activity in infected cells was measured by L929 cytotoxic assay, the highest expression level, 1.5 ´ 104 U/106 cells, was obtained at 76 h after infection. Western blot analysis of protein extracts from infected larvae showed that the virus-mediated TNF-a had immunoreactivity. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: TNF-a; Baculovirus system; Transfer vector; Gene expression
1. Introduction Tumor necrosis factor (TNF) is an antitumor protein found in the sera of animals and its cellular origin is macrophages (monocytes). The mature TNF form is secreted as a 157 amino acid protein after an unusually long, 76 amino acid, prepeptide sequence has been cleaved (Chai et al., 1993). TNF shows many biological activities, such as anti-tumor, anti-virus, immune regulation, and so on. The TNF cDNA has been expressed in Escherichia coli, monkey Cos-1 cells and yeast (Berent et al., 1986; Chai et al., 1993; Li et al., 1995). A baculovirus expression vector system, based on the Autographa californica nuclear polyhedrosis virus (AcNPV), is being increasingly used for the synthesis of heterologous proteins (Chatterji et al., 1996). Insect cells provide a suitable environment for post-translational modi®cations and folding of the protein product such that the foreign proteins synthesized are identical to their authentic counterparts in almost all respects (Miller, 1988; Luckow and Summers, 1988). Although there was a report about the expression of TNF-b in baculovirus expression vector system (Chai et al., 1993), little information regarding the TNF-a expressed in insect system is published in the available literature. Here
* Corresponding author. Tel.: +0086-020-8411-2299; fax: +0086-0208403-7249; e-mail: [email protected]
the synthesis of human TNF-a protein in Spodoptera frugiperda cells (Sf9) and Plusia agnata larvae infected with a genetically engineered baculovirus carrying the gene encoding the respective protein is investigated. 2. Methods Strains, plasmid and virus: Trichoplusia ni nuclear polyhedrosis virus (TnNPV) (NERC, Institute of Virology, Oxford, England) and its recombinant parent virus TnNPV-SVI± G DNA (gal OCC± ) containing the Syn XIV promoter (Wang et al., 1991); Spodoptera frugiperda (Sf9) cell were used in this study. The cell medium was Tc-100 containing 10% FBS. DNA manipulation and construction of recombinant plasmid: TNF expression plasmid PUC9-TNF1 (Berent et al., 1986) and baculovirus transfer vector pSXIVVI X3 without an initiation codon (Wang et al., 1991) were used for expression of TNF. E.Coli DH5a was used for propagation. Plasmid DNA manipulation, co-transfection, isolation, puri®cation and characterization of virus were carried out as described (Sambrook et al., 1989; Summers and Smith, 1987). Activity of TNF-a in infected cell: 1.5 ml portions of recombinant virus solutions were infected with Sf9 Cells (3 ´ 106 cell, MOI 10 pfu/cell), after 1 h the virus solutions were removed and 5 ml of medium Tc-100 containing 10% FBS was added. The supernatant of
0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 3 2 - 2
300
X.-W. Zhang et al. / Bioresource Technology 70 (1999) 299±301
infected-cell lysates was harvested after 24, 48, 72, 96 and 120 h of post-infection, respectively. The tumoricidal activity of TNF-a was assayed by measuring its cytotoxic activity against mouse L929 cells (Chai et al., 1993). Western blot analysis of infected larvae protein extracts: Fourth instar of Plusia agnata larvae were fed with a diet coated with recombinant virus polyhedrin; the sixth instar of larvae were harvested 4 days postinfection. Total cell extracts of virus-infected caterpillars were prepared and used for further analysis (Jha et al., 1992). Western blot analysis was carried out as described (Sambrook et al., 1989). Western blot of larvae protein extracts separated by SDS-PAGE (using slab gels with 15% polyacrylamide separating gel and 4% stacking gel) was probed with anti-human TNF monoclonal antibody E6 ,T5 (Chinese Academy of Military Medical Sciences, Beijing, China) with a dilution of 1:10000, the membranes were incubated in rabbit antimouse IgG (alkaline phosphatase, Sigma) with a dilution of 1:500, according to the manufacturer's instruction. 3. Results and discussion Construction of transfer vector carrying TNF-a cDNA: The EcoRI fragment carrying the cDNA encoding TNF-a from PUC9 (Berent et al., 1986) was cloned in the EcoRI site of transfer vector plasmid pSXIVVI X3 (5.8 kb, Wang et al., 1991), The ligation mix was digested with EcoRI and transformed (E.coli DH5a), thus re-plasmid pSXIVVI X3-TNF-a (6.3 kb) was generated, and the correct orientation for TNF-a cDNA inserted was con®rmed by detailed analysis (data not shown). The partial physical map of the transfer vector, pSXIVVI X3-TNF-a, used for generating recombinant virus, is illustrated in Fig. 1. Construction and characterization of recombinant baculovirus containing TNF-a gene: For constructing the recombinant baculovirus, Sf9 insect cells (NERC, Institute of Virology, Oxford, England) were co-transfected with the recombinant plasmid and TnNPV-SVI± G
Fig. 1. Partial physical map of the transfer vector pSXIVVI X3-TNFa showing the composition of the promoter cassettes and the direction of transcription of the genes encoding TNF-a. Relevant restriction sites, used in checking the orientation of the insert with respect to the promoter, are indicated. Polh±Polyhedrin promoter, XIV±XIV promoter.
DNA, a recombinant virus (gal OCCÿ , Wang et al., 1991). The recombinant baculovirus designated TnNPV-TNF-a-OCC , which contains TNF-a cDNA and can also form polyhedrin, was obtained by plaque puri®cation (O'Reilly et al., 1992; Summers and Smith, 1987). Enzyme digestion identi®cation revealed that EcoRI+SacI digested recombinant baculovirus had a 0.5 kb band, which was not visible in the two enzymes digestion map of the parent virus (data not shown). Expression of TNF-a cDNA in insect cells and larvae: Sf9 cells were infected with recombinant baculovirus TnNPV-TNF-a-OCC . SDS-PAGE and Western blot analysis revealed that TNF-a could be detected in the infected cell lysates 24 h post-infection, and the following empirical equation could be used to ®t the experimental data from three experiments for describing the relationship between the activity of TNF-a (A, ´ 104 u/ 106 cells) and the time after infection (t, h): 1 logA ÿ0:00096t2 0:1450t ÿ 1:3275: A satisfactory result was found between the experimental data and the calculated values with the average relative error 4.28% (Fig. 2). According to Eq. (1), the activity of TNF-a reached the maximum value 1.4972 ´ 104 u/106 cells at 75.9003 h after infection. Densitometric scanning of SDS-PAGE revealed that 38% of the total Coomassie brilliant blue-stained protein of the infected cells was represented by the recombinant TNF-a (data not shown). P. agnata larvae were infected with the recombinant baculovirus TnNPV-TNF-a-OCC simply by mixing the recombinant baculovirus into the insect diet. Larval protein extracts were analyzed by SDS-PAGE and western blot, the result showed that TNF-a could also be expressed in the larvae, with the molecular weight of 17 kDa, reacted with anti-human TNF-a monoclonal
Fig. 2. Time course of recombinant baculovirus TnNPV-TNF-aOCC expressing TNF-a in Sf9 cells. The open circle for the experimental, the ®lled circle for the calculated values.
X.-W. Zhang et al. / Bioresource Technology 70 (1999) 299±301
301
erpillars, this is valuable for the large-scale production of such a protein with low costs. References
Fig. 3. Western blot of TNF-a expression using recombinant baculovirus in larvae, (A) Larvae infected with TnNPV for 72 h postinfection. (B) Larvae infected with recombinant virus TnNPV-TNF-a-OCC 4 days postinfection. (M) Low molecular weight markers.
antibody (Fig. 3). This proved that the recombinant TNF-a was similar to the native. A large majority of the recombinant virus-infected larvae did not molt into pupae, and remained in the larval stage till their death due to the arrest of metamorphosis caused by the viral egt gene (Jha et al., 1992). The prolonged life span of infected larvae by 5±6 days is of special relevance for harvesting of recombinant proteins from larvae. Most, if not all, foreign proteins synthesized during the larval stage naturally have sucient time to undergo proper post-translational modi®cations. Results presented above demonstrate that the cDNA encoding human TNF-a can be expressed successfully in an occluded recombinant baculovirus expression system with the control of synthetic and XIV promoters. Specially, the recombinant TNF-a can be produced in cat-
Berent, S.L., Torczynski, R.M., Bollon, A.P., 1986. Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes. Nucleic Acids Res. 14, 8997±9013. Chai, H., Vasudevan, S.G., Porter, A.G., Chua, K.L., Oh, S., Yap, M., 1993. Glycosylation and high-level secret of hTNF-beta in recombinant baculovirus-infected insect cells . Biotechnol. Appl. Biochem. 18 (3), 259±273. Chatterji, U., Ahmad, R., Venkaiah, B., Hasnain, S.E., 1996. A recombination-ecient baculovirus vector for simultaneous expression of multiple genes. Gene 171, 209±213. Jha, P.K., Pal, R., Nakhai, B., Sridhar, P., Hasnain, S.E., 1992. Simultaneous synthesis of enzymatically active luciferase and biologically active-beta subunit of human chorionic gonadotropin in caterpillars infected with a recombinant baculovirus. FEBS Lett. 310, 148±152. Li, X.P., Li, Y., Liu, J.P., Wu, M., 1995. Cloning and expression of human TNF-alpha gene. Chinese J. Biotechnol. 11 (1), 73±76. Luckow, V.A., Summers, M.D., 1988. Trends in the development of baculovirus vectors. Biotechnology 6, 45±55. Miller, L.K., 1988. Baculovirus as gene expression vectors. Annu. Rev. Microbiol. 42, 177±199. O'Reilly, D.R., Miller, L.K., Luckow, V.A., 1992. Baculovirus expression vectors±A Laboratory Manual. W.H. Freman and company, New York. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, 2nd ed. CSHL Press, Cold Spring Harbor, New York. Summers, M.D., Smith, G.E., 1987. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agric. Exp. Stn. Bull. 1555, College Station, TX, USA. Wang, X., Ooi, B.G., Miller, L.K., 1991. Baculovirus vectors for multiple gene expression and for occluded virus production. Gene 100, 131±137.
Short communication
Expression of recombinant human tumor necrosis factor-a in a baculovirus expression system Xue-Wu Zhang*, Tao Sun, Xiang-Yue Zeng, Xin Liu, De-Xiang Gu Food Engineering Research Center of State Education Commission, Zhongshan University, Guangzhou 510275, People's Republic of China Received 17 September 1998; received in revised form 2 February 1999; accepted 10 February 1999
Abstract A cDNA fragment coding human tumor necrosis factor-alpha (TNF-a) was inserted into the vector pSXIVVI X3 with the control of Syn XIV promoter. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). Cells infected with recombinant virus synthesized TNF-a protein at a level of about 38% of total cellular protein. TNF-a activity in infected cells was measured by L929 cytotoxic assay, the highest expression level, 1.5 ´ 104 U/106 cells, was obtained at 76 h after infection. Western blot analysis of protein extracts from infected larvae showed that the virus-mediated TNF-a had immunoreactivity. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: TNF-a; Baculovirus system; Transfer vector; Gene expression
1. Introduction Tumor necrosis factor (TNF) is an antitumor protein found in the sera of animals and its cellular origin is macrophages (monocytes). The mature TNF form is secreted as a 157 amino acid protein after an unusually long, 76 amino acid, prepeptide sequence has been cleaved (Chai et al., 1993). TNF shows many biological activities, such as anti-tumor, anti-virus, immune regulation, and so on. The TNF cDNA has been expressed in Escherichia coli, monkey Cos-1 cells and yeast (Berent et al., 1986; Chai et al., 1993; Li et al., 1995). A baculovirus expression vector system, based on the Autographa californica nuclear polyhedrosis virus (AcNPV), is being increasingly used for the synthesis of heterologous proteins (Chatterji et al., 1996). Insect cells provide a suitable environment for post-translational modi®cations and folding of the protein product such that the foreign proteins synthesized are identical to their authentic counterparts in almost all respects (Miller, 1988; Luckow and Summers, 1988). Although there was a report about the expression of TNF-b in baculovirus expression vector system (Chai et al., 1993), little information regarding the TNF-a expressed in insect system is published in the available literature. Here
* Corresponding author. Tel.: +0086-020-8411-2299; fax: +0086-0208403-7249; e-mail: [email protected]
the synthesis of human TNF-a protein in Spodoptera frugiperda cells (Sf9) and Plusia agnata larvae infected with a genetically engineered baculovirus carrying the gene encoding the respective protein is investigated. 2. Methods Strains, plasmid and virus: Trichoplusia ni nuclear polyhedrosis virus (TnNPV) (NERC, Institute of Virology, Oxford, England) and its recombinant parent virus TnNPV-SVI± G DNA (gal OCC± ) containing the Syn XIV promoter (Wang et al., 1991); Spodoptera frugiperda (Sf9) cell were used in this study. The cell medium was Tc-100 containing 10% FBS. DNA manipulation and construction of recombinant plasmid: TNF expression plasmid PUC9-TNF1 (Berent et al., 1986) and baculovirus transfer vector pSXIVVI X3 without an initiation codon (Wang et al., 1991) were used for expression of TNF. E.Coli DH5a was used for propagation. Plasmid DNA manipulation, co-transfection, isolation, puri®cation and characterization of virus were carried out as described (Sambrook et al., 1989; Summers and Smith, 1987). Activity of TNF-a in infected cell: 1.5 ml portions of recombinant virus solutions were infected with Sf9 Cells (3 ´ 106 cell, MOI 10 pfu/cell), after 1 h the virus solutions were removed and 5 ml of medium Tc-100 containing 10% FBS was added. The supernatant of
0960-8524/99/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 3 2 - 2
300
X.-W. Zhang et al. / Bioresource Technology 70 (1999) 299±301
infected-cell lysates was harvested after 24, 48, 72, 96 and 120 h of post-infection, respectively. The tumoricidal activity of TNF-a was assayed by measuring its cytotoxic activity against mouse L929 cells (Chai et al., 1993). Western blot analysis of infected larvae protein extracts: Fourth instar of Plusia agnata larvae were fed with a diet coated with recombinant virus polyhedrin; the sixth instar of larvae were harvested 4 days postinfection. Total cell extracts of virus-infected caterpillars were prepared and used for further analysis (Jha et al., 1992). Western blot analysis was carried out as described (Sambrook et al., 1989). Western blot of larvae protein extracts separated by SDS-PAGE (using slab gels with 15% polyacrylamide separating gel and 4% stacking gel) was probed with anti-human TNF monoclonal antibody E6 ,T5 (Chinese Academy of Military Medical Sciences, Beijing, China) with a dilution of 1:10000, the membranes were incubated in rabbit antimouse IgG (alkaline phosphatase, Sigma) with a dilution of 1:500, according to the manufacturer's instruction. 3. Results and discussion Construction of transfer vector carrying TNF-a cDNA: The EcoRI fragment carrying the cDNA encoding TNF-a from PUC9 (Berent et al., 1986) was cloned in the EcoRI site of transfer vector plasmid pSXIVVI X3 (5.8 kb, Wang et al., 1991), The ligation mix was digested with EcoRI and transformed (E.coli DH5a), thus re-plasmid pSXIVVI X3-TNF-a (6.3 kb) was generated, and the correct orientation for TNF-a cDNA inserted was con®rmed by detailed analysis (data not shown). The partial physical map of the transfer vector, pSXIVVI X3-TNF-a, used for generating recombinant virus, is illustrated in Fig. 1. Construction and characterization of recombinant baculovirus containing TNF-a gene: For constructing the recombinant baculovirus, Sf9 insect cells (NERC, Institute of Virology, Oxford, England) were co-transfected with the recombinant plasmid and TnNPV-SVI± G
Fig. 1. Partial physical map of the transfer vector pSXIVVI X3-TNFa showing the composition of the promoter cassettes and the direction of transcription of the genes encoding TNF-a. Relevant restriction sites, used in checking the orientation of the insert with respect to the promoter, are indicated. Polh±Polyhedrin promoter, XIV±XIV promoter.
DNA, a recombinant virus (gal OCCÿ , Wang et al., 1991). The recombinant baculovirus designated TnNPV-TNF-a-OCC , which contains TNF-a cDNA and can also form polyhedrin, was obtained by plaque puri®cation (O'Reilly et al., 1992; Summers and Smith, 1987). Enzyme digestion identi®cation revealed that EcoRI+SacI digested recombinant baculovirus had a 0.5 kb band, which was not visible in the two enzymes digestion map of the parent virus (data not shown). Expression of TNF-a cDNA in insect cells and larvae: Sf9 cells were infected with recombinant baculovirus TnNPV-TNF-a-OCC . SDS-PAGE and Western blot analysis revealed that TNF-a could be detected in the infected cell lysates 24 h post-infection, and the following empirical equation could be used to ®t the experimental data from three experiments for describing the relationship between the activity of TNF-a (A, ´ 104 u/ 106 cells) and the time after infection (t, h): 1 logA ÿ0:00096t2 0:1450t ÿ 1:3275: A satisfactory result was found between the experimental data and the calculated values with the average relative error 4.28% (Fig. 2). According to Eq. (1), the activity of TNF-a reached the maximum value 1.4972 ´ 104 u/106 cells at 75.9003 h after infection. Densitometric scanning of SDS-PAGE revealed that 38% of the total Coomassie brilliant blue-stained protein of the infected cells was represented by the recombinant TNF-a (data not shown). P. agnata larvae were infected with the recombinant baculovirus TnNPV-TNF-a-OCC simply by mixing the recombinant baculovirus into the insect diet. Larval protein extracts were analyzed by SDS-PAGE and western blot, the result showed that TNF-a could also be expressed in the larvae, with the molecular weight of 17 kDa, reacted with anti-human TNF-a monoclonal
Fig. 2. Time course of recombinant baculovirus TnNPV-TNF-aOCC expressing TNF-a in Sf9 cells. The open circle for the experimental, the ®lled circle for the calculated values.
X.-W. Zhang et al. / Bioresource Technology 70 (1999) 299±301
301
erpillars, this is valuable for the large-scale production of such a protein with low costs. References
Fig. 3. Western blot of TNF-a expression using recombinant baculovirus in larvae, (A) Larvae infected with TnNPV for 72 h postinfection. (B) Larvae infected with recombinant virus TnNPV-TNF-a-OCC 4 days postinfection. (M) Low molecular weight markers.
antibody (Fig. 3). This proved that the recombinant TNF-a was similar to the native. A large majority of the recombinant virus-infected larvae did not molt into pupae, and remained in the larval stage till their death due to the arrest of metamorphosis caused by the viral egt gene (Jha et al., 1992). The prolonged life span of infected larvae by 5±6 days is of special relevance for harvesting of recombinant proteins from larvae. Most, if not all, foreign proteins synthesized during the larval stage naturally have sucient time to undergo proper post-translational modi®cations. Results presented above demonstrate that the cDNA encoding human TNF-a can be expressed successfully in an occluded recombinant baculovirus expression system with the control of synthetic and XIV promoters. Specially, the recombinant TNF-a can be produced in cat-
Berent, S.L., Torczynski, R.M., Bollon, A.P., 1986. Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes. Nucleic Acids Res. 14, 8997±9013. Chai, H., Vasudevan, S.G., Porter, A.G., Chua, K.L., Oh, S., Yap, M., 1993. Glycosylation and high-level secret of hTNF-beta in recombinant baculovirus-infected insect cells . Biotechnol. Appl. Biochem. 18 (3), 259±273. Chatterji, U., Ahmad, R., Venkaiah, B., Hasnain, S.E., 1996. A recombination-ecient baculovirus vector for simultaneous expression of multiple genes. Gene 171, 209±213. Jha, P.K., Pal, R., Nakhai, B., Sridhar, P., Hasnain, S.E., 1992. Simultaneous synthesis of enzymatically active luciferase and biologically active-beta subunit of human chorionic gonadotropin in caterpillars infected with a recombinant baculovirus. FEBS Lett. 310, 148±152. Li, X.P., Li, Y., Liu, J.P., Wu, M., 1995. Cloning and expression of human TNF-alpha gene. Chinese J. Biotechnol. 11 (1), 73±76. Luckow, V.A., Summers, M.D., 1988. Trends in the development of baculovirus vectors. Biotechnology 6, 45±55. Miller, L.K., 1988. Baculovirus as gene expression vectors. Annu. Rev. Microbiol. 42, 177±199. O'Reilly, D.R., Miller, L.K., Luckow, V.A., 1992. Baculovirus expression vectors±A Laboratory Manual. W.H. Freman and company, New York. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, 2nd ed. CSHL Press, Cold Spring Harbor, New York. Summers, M.D., Smith, G.E., 1987. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agric. Exp. Stn. Bull. 1555, College Station, TX, USA. Wang, X., Ooi, B.G., Miller, L.K., 1991. Baculovirus vectors for multiple gene expression and for occluded virus production. Gene 100, 131±137.