Biotechnol. Appl. Biochem. (2003) 37, 109–113 (Printed in Great Britain) 109

Construction of a new tumour necrosis factor fusion-proteinexpression vector for high-level expression of heterologousgenes in Escherichia coli

Wei Han, Yingqi Zhang1, Zhen Yan and Jihong Shi

Biotechnology Center of The Fourth Military Medical University, Chang Le Xi Lu 17g, Xi’an, Shaanxi 710032,People’s Republic of China

We report the construction and application of a newfusion-protein expression plasmid (TNFHis) forEscherichia coli. The plasmid contains both PR and PL

promoters and is optimized to allow a higher level ofexpression of mature coding sequences. It also containsa six-histidine tag for convenient purification as well asthrombin and hydroxylamine recognition sites forcleaving heterologous protein. The potential use ofthis expression vector is demonstrated by comparingthe expression levels of human tumour necrosis factor(TNF), interferon, interleukin 11, colony-forming fac-tor, osteoprotegrin and interleukin 2 in E. coli. Fur-thermore, all expressed TNF fusion proteins can bedetected by anti-TNFα antibody or by specific anti-bodies and purified by Ni2+-nitrilotriacetate beads. Theexpressed TNF fusion proteins can be cleaved byhydroxylamine.

Introduction

Escherichia coli is a high-level expression host and has beenwidely used in both molecular biology laboratories and thepharmaceutical industry. Many expression vectors for E. colihave been constructed and well characterized. Some ofthem are very efficient and have been used successfully formammalian genes expression.

Currently, there are two types of expression vector inE. coli for expressing mammalian genes. One is the non-fusion expression vector and the other is the fusionexpression vector. The former is easier to use in the puri-fication process if an antibody is available for affinitypurification of the expressed protein. However, certaingenes are not expressed well or even not expressed at all innon-fusion expression vectors. In such cases, the only choicefor expressing genes is to use a fusion expression vector.Several kinds of fusion expression vectors have beenconstructed, characterized and commercialized in thepast 10 years. The fusion partners include glutathioneS-transferase (‘GST’) [1], maltose-binding protein, staphy-lococcal protein A and thioredoxin [2]. The most greatestadvantage of fusion expression vectors is that the inserted

genes can usually be expressed well. The major shortcomingof current fusion expression vectors is that chemicals, suchas isopropyl β-D-thiogalactoside (‘ IPTG’), are still necessaryfor the induction of fusion protein expression, which can-not meet the demand for recombinant protein productionon the scale required by the pharmaceutical industry.

We have previously constructed an expression vector,pBVTNF [3], which was derived from the pBV220 vector andcontained the PR and PL promoters, the cIts857 gene and twostrong transcription terminators [4]. In the present reportwe describe a new tumour necrosis factor (TNF) fusion-protein expression vector, TNFHis, which was modifiedfrom pBVTNF.

Materials and methods

Enzymes and plasmidsRestriction enzymes and T4 ligase were purchased fromGibco BRL. Plasmids used for different constructions wereas follows: pGEM3Zf−, pBVTNF and pBV220. All cloningprocedures were performed following standard protocols[5]. All antibodies were purchased from Santa Cruz Bio-technology.

Construction of the TNFHis expression vectorThe original pBVTNF expression vector is of the non-fusiontype. We performed several modifications on the vector.We designed two synthetic DNA fragments. One containeda start codon and the sequences of the first two amino acidsof TNF as well as a six-histidine tag. The other includedsequences for thrombin and hydroxylamine recognition sites

Key words : affinity purification, histidine tag, PL promoter, PR promoter.Abbreviations used : TNF, tumour necrosis factor ; IFN, interferon ; IL-11,

interleukin 11 ; CSF, colony-forming factor ; OPG, osteoprotegrin ;IL-2, interleukin 2 ; Ni-NTA, Ni2+-nitrilotriacetate.

1 To whom correspondence should be addressed (e-mailZhangyqo!fmmu.edu.cn).

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110 W. Han and others

Figure 1 Map of the TNFHis expression vector

Construction details are available from the corresponding author on request.

as well as multiple cloning sites. The former fragment wasinserted into the 5« end of TNF and the latter was ligated tothe 3« end of TNF of pBVTNF. A map of the final constructionplasmid is shown in Figure 1. The plasmid was designatedTNFHis.

Expression of heterologous genes in the TNFHisvectorHuman interferon (IFN), TNF, interleukin 11 (IL-11), colony-forming factor (CSF), osteoprotegrin (OPG) and interleukin2 (IL-2) genes were ligated into the EcoRI and BamHI sites ofthe TNFHis plasmid. Luria–Bertani medium was inoculatedand cultures were grown overnight with aeration at 30 °C.When the D600 reached 0.8, the temperature was changed to42 °C and cultures were further incubated for 4–5 h. Thenthe cells were harvested by centrifugation for 10 min at8000 g at 4 °C and denatured by boiling at 100 °C for 5 minbefore SDS}PAGE analysis [6]. 12% polyacrylamide gelswere used throughout the study.

Western-blot analysisSDS}PAGE was performed following standard procedures.Total protein (10 µg) from each bacterial pellet sample wasdenatured by boiling at 100 °C for 5 min and loaded on thegels. Electrophoresis was performed for 2 h at 120 mV. Gelswere blotted and the membrane was blocked for 1 h atroom temperature in 2% BSA. Anti-TNFα antibody wasused at a dilution of 1 :100. The protein bands were visualizedusing the ProtoBlot Western Blot AP System (Promega).

DNA sequencingSequencing of DNA fragments generated by PCR amplifi-cation was conducted by automatic DNA sequencing on

double-stranded DNA templates with an Applied Bio-systems 377 sequencer.

Purification of TNF fusion protein by affinitychromatographyThe cells were resuspended in 10 vol. of buffer A (100 mMNaH2PO4}10 mM Tris}HCl, pH 8.0). After adding 1 mg}mllysozyme, the cells were stirred for 1 h at room temperature.Buffer B (10 ml ; 6 M guanidine}HCl, 100 mM NaH2PO4 and10 mM Tris}HCl, pH 8.0) was added and stirred for another30 min at room temperature. Samples were centrifuged for15 min at 11600 g at 4 °C. Then 8 ml of a 50% slurry of Ni2+-nitrilotriacetate (Ni-NTA)–agarose was added and stirred atroom temperature for 60 min. The mixture was loaded intothe column and washed with 10 vol. of buffer B, 5 vol. ofbuffer C (8 M urea, 100 mM NaH2PO4 and 10 mM Tris}HCl,pH 8.0) followed by 15–20 vol. of buffer D (8 M urea and100 mM NaH2PO4, pH 6.3) until the A280 of the washedsolution dropped below 0.01. Finally, TNF fusion proteinswere eluted with 20 ml of buffer E (8 M urea, 100 mMNaH2PO4 and 10 mM Tris}HCl, pH 5.9) and analysed bySDS}PAGE.

Cleavage of TNFHis fusion proteins by hydroxylamineHydroxylamine (8 M) was added to the supernatant ofpartially purified fusion proteins (the final concentrationof hydroxylamine was 1 M) and incubated at 35 °C for 15 h.The supernatant was analysed by SDS}PAGE after cleavagewas complete.

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A new vector for high-level expression in Escherichia coli 111

Results and discussion

Comparison of expression levels between TNFHis,pBVTNF1 and pBVTNFH expression vectorsThe expression level of TNF in TNFHis vector is the mostcrucial for its potential usage. Therefore we first comparedthe expression levels of TNF among TNFHis, pBVTNF andpBVTNFH (in which only a six-histidine tag sequence wasfused to the 5« end of TNF) vectors by SDS}PAGE. Theresult (Figure 2) showed that the expression level of TNF inTNFHis vector was about 30% of the total E. coli cellularprotein content and was similar to the levels of TNF in thepBVTNF or pBVTNFH vectors.

Another point we were concerned with was that thesynthetic six-histidine sequence might interrupt expressionof TNF because the sequence is a heterologous sequence forthe TNF gene. To observe an effect of the synthetic six-histidine sequence on the expression levels of TNF, weanalysed expression of TNF in pBVTNFH by SDS}PAGE. Asshown in Figure 2, there was no obvious difference in termsof TNF expression among the pBVTNFH, pBVTNF andTNFHis vectors.

Induction and expression of human cytokine genes inTNFHis vectorTo investigate whether the TNFHis vector could expressthe fused proteins, six human cytokine genes, IFN, TNF,IL-11, CSF, OPG and IL-2, were inserted into TNFHis in thematched reading frame. After recombinant bacteria wereincubated at 42 °C, expression levels of the different TNFfusion proteins were analysed by SDS}PAGE (Figure 3).

The amounts of all expressed TNFHis fusion proteinswere identified with a densitometer. Scanning densitometry

Figure 2 Expression analysis of pBVTNF, pBVTNFH and TNFHis

Lane 1, molecular-mass markers ; lane 2, sample taken before induction ; lane 3,42 °C-induced pBVTNF ; lane 4, 42 °C-induced pBVTNFH; lanes 5and 6, 42 °C-induced TNFHis. The SDS/PAGE gel was stained with CoomassieBrilliant Blue R250. Arrows show expression of TNF from each vector ;TNFHis, THFHis vector ; TNFH, pBVTNFH vector ; TNF, pBVTNF vector.

Figure 3 Expression analysis of different TNF fusion proteins

Top panel : lane 1, molecular-mass markers ; lane 2, induced TNFHis ; lane 3,uninduced TNFHis-TNF; lane 4, induced TNFHis-TNF; lane 5, uninducedTNFHis-IL-11 ; lane 6, induced TNFHis-IL-11 ; lane 7, uninduced TNFHis-IFN;lane 8, induced TNFHis-IFN. Bottom panel : lane 1, molecular-mass markers ;lane 2, uninduced TNFHis-OPG; lane 3, induced TNFHis-OPG; lane 4,uninduced TNFHis-CSF ; lane 5, induced TNFHis-CSF ; lane 6, uninducedTNFHis-IL-2 ; lane 7, induced TNFHis-IL-2. Stained was with Coomassie BrilliantBlue R250.

indicated that expression of the six fusion proteins rangedfrom 13 to 43% of the total E. coli cellular protein content.

Of those human genes, IL-11 and IFN almost cannot beexpressed by non-fusion expression vectors in E. coli.However, the levels of expression of both genes wereincreased dramatically in the TNFHis vector. This suggestedthat all heterologous genes, which are difficult to express bynon-fusion vectors in E. coli, could possibly be expressedby the TNFHis vector.

Identification of expression of heterologous genes byWestern blottingTNF fusion proteins can easily be identified by blotting withthe anti-TNFα antibody. The immunoblot assay proved thatTNFHis-IFN, TNFHis-TNF and TNFHis-IL-11 proteins weredetected with the anti-TNFα antibody (Figure 4, top panel).It also demonstrated that the expression of all TNF fusionproteins in the TNFHis vector could easily be detected usingthe anti-TNFα antibody.

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112 W. Han and others

Figure 4 Identification of TNF fusion proteins by Western blot

Top panel : lane 1, induced TNFHis-IFN; lane 2, uninduced TNFHis-IFN; lane 3,induced TNFHis-IL-11 ; lane 4, uninduced TNFHis-IL-11 ; lane 5, inducedTNFHis-TNF; lane 6, uninduced TNFHis-TNF; lane 7, induced TNFHis. Bottompanel : lane 1, uninduced TNFHis-OPG; lane 2, induced TNFHis-OPG (primaryantibody is anti-OPG antibody) ; lane 3, uninduced TNFHis-CSF ; lane 4,induced TNFHis-CSF (primary antibody is anti-CSF antibody) ; lane 5, uninducedTNFHis-IL-2 ; lane 6, induced TNFHis-IL-2 (primary antibody is anti-IL-2).

In addition, we blotted the TNFHis-OPG, TNFHis-CSFand TNFHis-IL-2 proteins with anti-OPG, anti-CSF and anti-IL-2 antibodies respectively. All three proteins could beidentified by their specific antibodies (Figure 4, bottompanel).

Purification of TNFHis-IFN and TNFHis-TNFproteins by Ni-NTA–agarose affinity chromatographyA number of refined gene expression vectors take advantageof the high affinities of certain protein sequences (tags) fortheir substrates, cofactors and other ligands to achieve rapidpurification of proteins fused with such tags [7]. Six-histidineor ten-histidine tags have been used widely for purification ofmany fusion proteins [8,9]. We also fused a six-histidine tagsequence to the 5« end of TNF in the fusion expressionvector in an attempt to provide a convenient way to purifyTNF fusion proteins.

Figure 5 Purification of TNFHis-IFN with Ni-NTA–agarose chromato-graphy

Lane 1, TNFHis-TNF; lane 2, TNFHis-IFN. Gel was stained with CoomassieBrilliant Blue R250.

Figure 6 Cleavage of TNFHis-IFN, TNFHis-TNF and TNFHis-IL-11proteins by hydroxylamine

Lane 1, molecular-mass markers ; lane 2, TNFHis-IFN (before cleavage) ; lane 3,TNFHis-IFN (after cleavage) ; lane 4, TNFHis-IL-11 (before cleavage) ; lane5, TNFHis-IL-11 (after cleavage) ; lane 6, TNFHis-TNF (before cleavage) ;lane 7, TNFHis-TNF (after cleavage). Stained was with Coomassie BrilliantBlue R250.

Since TNFHis-IFN and TNFHis-TNF proteins are bothinsoluble, we purified them by Ni-NTA–agarose affinitychromatography under denaturing conditions [10]. Theresults of the affinity purification of TNFHis-IFN andTNFHis-TNF are shown in Figure 5. They indicated that it ispossible to purify TNF fusion proteins with one-step affinitychromatography.

Cleavage of TNFHis fusion proteinsAfter the TNFHis-IFN, TNFHis-TNF and TNFHis-IL-11proteins were partially purified and cleaved for 15 h withhydroxylamine, the supernatants of the proteins wereanalysed by SDS}PAGE. The results were shown in Figure 6.This showed that the expressed heterologous proteins in

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A new vector for high-level expression in Escherichia coli 113

the TNFHis vector can be cleaved away from the TNF fusionproteins by digestion with hydroxylamine.

In conclusion, we constructed a new TNF fusion-protein expression vector that contains the PR and PL

promoters. Six human cytokine genes were expressed in thisvector. The vector provides the possibility for easy puri-fication of TNF fusion proteins by one-step affinity chromato-graphy. Moreover, the expressed TNF fusion proteins canbe cleaved by hydroxylamine. This experimental evidencedemonstrates that the TNFHis expression vector is a usefultool for expression of heterologous genes.

We are grateful to Dr Liu Xuesong for providing the anti-TNF-α antibody.

References

1 Smith, D. B. and Johnson, K. S. (1988) Gene 67, 31–40

2 Olins, P. O. and Lee, S. C. (1993) Curr. Opin. Biotechnol. 4,

520–525

3 Yang, H., Zhang, Y. Q. and Li, F. M. (1997) J. Fourth Military

Medical Uni. 18, 221–224

4 Zhang, Z. Q., Yao, L. H. and Hou, Y. D. (1990) Chin. J. Virol. 6,

111–116

5 Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular

Cloning : a Laboratory Manual, 2nd edn, Cold Spring Harbor

Press, Cold Spring Harbor, NY

6 Leammli, U. K. (1970) Nature (London) 227, 680–683

7 Uhlen, M. and Mocks, T. (1990) Methods Enzymol. 185,

129–143

8 Hochuli, E. (1990) in Genetic Engineering, Principle and

Methods (Setlow, J. K., ed.), pp. 87–89, Plenum, New York

9 Van Dyke, M. W., Sirito, M. and Sawadogo, M. (1992) Gene

111, 99–104

10 Qiagen (2000) A Handbook for High-Level Expression and

Purification of 6xHis-Tagged Protein, The QIAexpressionist,

Qiagen, Valencia, CA

Received 13 August 2002}13 September 2002; accepted 16 October 2002

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