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Published online before print May 7, 2004, 10.1110/ps.04644504
Protein Science (2004), 13:1698-1703. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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FOR THE RECORD

Two-promoter vector is highly efficient for overproduction of protein complexes

Kyung-Jin Kim1, Han-Eol Kim, Kwang-Hoon Lee, Wondeok Han, Min-Ju Yi, Jinseok Jeong and Byung-Ha Oh

Center for Biomolecular Recognition and Division of Molecular and Life Science, Department of Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea

Reprint requests to: Byung-Ha Oh, Center for Biomolecular Recognition and Division of Molecular and Life Science, Department of Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea; e-mail: bhoh{at}postech.ac.kr; fax: 82-54-279-2199; or Kyung-Jin Kim, Pohang Accelerator Laboratory, Pohang, Kyungbuk, 790-784, Korea; e-mail: kkj{at}postech.ac.kr; fax: 82-54-2119-1599.

(RECEIVED January 19, 2004; FINAL REVISION February 19, 2004; ACCEPTED February 20, 2004)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes. The major problem of this method is the much lower expression of the second gene compared with that of the first gene next to the promoter. We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem. Compared with bicistronic vectors, corresponding two-promoter vectors yielded four to nine times larger amounts of the complexes between BCL-2 family proteins, BCL-XL:BAD, BCL-XL:BIM-S, and CED-9:EGL-1 in bacterial cells as a result of significantly increased expression of the second genes in a manner independent of the order of the target genes. With the two-promoter system, we produced two other complexes in large quantity suitable for extensive crystallization trial. The method does not accompany any technical disadvantages, and represents a significant improvement from the conventional method, which should enjoy wide application for the coexpression of binary or higher order protein complexes by extension.

Keywords: protein expression system; two-promoter; protein complex; apoptosis

1 Present address: Pohang Accelerator Laboratory, Pohang, Kyungbuk, 790-784, Korea. Back

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04644504.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Many proteins exert their functions through protein:protein interactions. Three-dimensional structures of protein complexes usually provide a wealth of information regarding the action mechanism of constituent proteins. Protein complexes may be obtained by simply mixing individually purified proteins. However, expression of some proteins requires a binding-partner protein for folding and stability. Two methods for heterologous coexpression of partner proteins have been commonly used: the bicistronic vector and the dual-vector systems. The bicistronic vector mimics the bacterial operon structure that consists of a cluster of genes under a single promoter. In theory, the expression of the second gene could be comparable to that of the first gene, because each translation sequence has the ribosome-binding site (RBS). In practice, much less expression of the second gene compared with that of the first gene has been commonly observed (Rucker et al. 1997). The dual-vector system, where two target genes are coexpressed from two different vectors, may overcome this problem. However, dominance of one vector over the other in the copy number is the major problem associated with this method, despite the use of vectors with a compatible origin of replication (Johnston et al. 2000).

By simply adding a promoter sequence in front of the second target gene on a bicistronic vector, the mRNA transcripts of the second target gene can be generated independently of the "read-through" transcript from the first promoter. Therefore, the resulting two-promoter vector should lead to an increased production of the protein encoded by the second target gene. Furthermore, because the two-promoter vector system is based on a single vector, it is free of the major problem associated with the dual-vector system. The two-promoter system was previously applied for the production of protein complex (McNally et al. 1988; Humphreys et al. 2002). However, systematic evaluation of the system and thorough comparison with conventional bicistronic system have not been reported.

We began to systemically evaluate the two-promoter vector system for the production of three different binary complexes between B-cell lymphoma 2 (BCL-2) family proteins in Escherichia coli, which are the fundamental regulators of apoptosis pathways (Cory and Adams 2002). The family comprises both proapoptotic and antiapoptotic members (Opferman and Korsmeyer 2003). The antiapoptotic members include BCL-XL, BCL-W, and CED-9, while the proapoptotic members includes BAD, BIM-S, EGL-1, and BMF. The antiapoptotic members antagonize the proapoptotic members by binding to them (Opferman and Korsmeyer 2003). Using two-promoter vectors, we obtained three other protein complexes in large quantity, all of which required coexpression of two binding partner proteins for the solubility or stability of one of the pair.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
Each of the two genes coding for BCL-XL (residues 1–196) and CED-9 (residues 68–244, C107S/C135A/C164S; Woo et al. 2003) was cloned into the pPosKJ vector, while each of the full-length genes coding for BAD, BIM-S, and EGL-1 was cloned into the pET30a vector. The pPosKJ vector was designed to express a target gene as a fusion protein with (His)6-tagged Vitreoscilla hemoglobin (VHb) at the N terminus, which can be cleaved off by TEV pro-tease (Fig. 1AGo). The red color associated with the heme group of VHb aids in the purification of a protein fused to it (Park et al. 2003). The pET30a vector was used for producing the target proteins without a tag (Fig. 1AGo). When these proteins were expressed individually in E. coli, VHb-fused BCL-XL and CED-9 were highly expressed in soluble forms and stable. However, the proapoptotic proteins were problematic. BAD was highly expressed in soluble form, but degraded completely within several days at 4°C. BIM-S was expressed but degraded into several pieces in E. coli cells, and the expression of EGL-1 was not detected (Woo et al. 2003). Endogenous BAD is held on check by sequestration to 14-3-3 protein (Zha et al. 1996; Datta et al. 1997), and BIM-S could be expressed stably in cultured cell lines only when BCL-2 or a functional homolog was coexpressed at high levels (O’Connor et al. 1998; Puthalakath et al. 1999). EGL-1 requires the presence of CED-9 for expression in human cells and in in vitro translation (del Peso et al. 1998). We hypothesized that these proteins require coexpression of an interacting protein to remain stable. The vectors derived from pPosKJ and pET30a were used to construct the bicistronic and two-promoter vectors to produce the VHb-BCL-XL:BAD, VHb-BCL-XL:BIM-S, and VHb-CED-9:EGL-1 complexes. In the both vector sets, VHb-BCL-XL and VHb-CED-9 were under T5 promoter and pre-ceded the second genes BAD, BIM-S, or EGL-1. In the two-promoter vectors, the second genes were under T7 promoter (Fig. 1AGo). BAD, BIM-S, and EGL-1 expressed as a complex with the partner protein from the bicistronic or the two-promoter vectors remained stable throughout protein purification and during a prolonged storage at 4°C. The amount of the VHb-BCL-XL:BAD, VHb-BCL-XL:BIM-S, and VHb-CED-9:EGL-1 complexes obtained with the bicistronic vectors from 1-L culture were 2.1, 0.5, and 2.8 mg, respectively. In contrast, 18, 2.2, and 16 mg of the respective complexes were obtained with the two-promoter vectors, corresponding to four to nine times higher yield compared with the bicistronic vector system (Fig. 1B,CGo). In the both systems, VHb-BCL-XL and VHb-CED-9 were produced in excess of the partner protein. Therefore, the increased production of the complexes resulted from the increased expression of the second genes, as observed on SDS-PAGE gels (Fig. 1BGo). This must be due to the generation of the mRNA transcripts of the second gene in addition to the read-through transcripts from the first promoter. After removal of the (His)6-tagged VHb by TEV protease, 8.5, 1.1, and 7.5 mg of the BCL-XL:BAD, BCL-XL:BIM-S, and CED-9:EGL-1 complexes were obtained, respectively, which exhibited greater than 95% purity on a SDS-PAGE gel (Fig. 2AGo). The purified complexes must be properly folded, because the circular dichroism (CD) spectra of these samples exhibited a high content of {alpha}-helices (Fig. 2BGo), as expected from the all {alpha}-helical structure of BCL-XL (Muchmore et al. 1996) and CED-9 (Woo et al. 2003). By using this method, we were able to obtain other complexes between the BCL-2 family proteins, BCL-XL:BMF and BCL-W:BAD (Fig. 2AGo), in milligram quantity (>7 mg) per 1-L culture.





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  		Figure 1. Vector construction and protein overproduction. (A) Construction of the bicistronic and two-promoter vectors. The vectors used for producing the BCL-XL:BAD complex is presented as a representative. T7, T5, and TEV denote T7 promoter, T5 promoter, and TEV protease cleavage site, respectively. The lines with double-arrows indicate the amplified DNA fragments used to produce the bicistronic or the two-promoter expression vector. (B) SDS-PAGE analysis. The production levels of the BCL-XL:BAD, BCL-XL:BIM-S, and CED-9:EGL-1 complexes were analyzed. Cleared cell lysate (C) and supernatant of the cell lysate (S) were loaded on a denaturing gel. Protein samples partially purified using a Ni-NTA column are denoted by "N." (C) Quantitative representation of protein production. "Total expressed protein" indicates the partially purified proteins from a Ni-NTA column as in b, which was the mixture of VHb-BCL-XL or VHb-CED-9 and the protein in complex with the binding partner. "Protein complex" indicates the purified complexes prepared by removing the excessive uncomplexed VHb-BCL-XL or VHb-CED-9 from the N fraction using an anion exchange column and, when needed, a Superdex G75 gel filtration column. "BC" and "TP" indicate the bicistronic and the two-promoter expression system, respectively. All data represent means ± standard deviation (error bars) of three separate experiments.

 



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  		Figure 2. SDS-PAGE and CD spectra of purified protein complexes. (A) SDS-PAGE analysis. The five different complexes of the BCL-2 family proteins were all produced using two-promoter vectors and purified. Although CED-9 appears in excess of EGL-1, the sample exhibited only one band corresponding to the CED-9:EGL-1 complex on a native gel. (B) CD spectra. The purified BCL-XL:BIM-S and CED-9:EGL-1 complexes exhibited typical spectra of {alpha}-helical proteins. The other purified protein complexes showed similar CD spectra.

 
In using mammalian bicistronic expression vectors, it has been observed that the expression of the internal ribosome entry site-dependent second gene is significantly lower than that of the cap-dependent first gene (Sugimoto et al. 1995; Mizuguchi et al. 2000), which is analogous to the pattern of the gene expression from bicistronic vectors in E. coli. To overcome the problem, two-promoter expression vectors were designed and successfully used to express two target genes in mammalian cells (Yu et al. 2003). In this experiment, the expression of the two genes varied considerably depending on the promoters used (Yu et al. 2003). To test whether different choices of promoters could affect the expression levels of two target genes in E. coli, we constructed two-promoter vectors with the promoter combinations of T7/T7 or Trc/T7 for the BCL-XL/BAD–(His)6 or (His)6–BCL-XL/BAD, as the first gene/second gene, respectively (Fig. 3Go). The expressions of BAD from these two-promoter vectors and the vector having the T5/T7 combination for VHb-BCL-XL/BAD (Fig. 1AGo) were nearly the same (data not shown), demonstrating that the choice of the promoter for the first target gene does not affect the expression of the second target gene BAD under the common T7 promoter. The expression levels of BCL-XL (after removing the attached tag) from the two-promoter vectors were different. However, it is not due to the difference in the promoter combination but due to the differences in the promoter and tagging sequence in front of it (VHb-BCL-XL > (His)6–BCL-XL > BCL-XL), because the expression level of BCL-XL was directly proportional to that of BCL-XL from the single promoter vector containing the same promoter and tagging sequence (data not shown). The observed compatibility of these most commonly used promoters for protein production in E. coli indicates that many commercially available vectors can be freely chosen for the construction of two-promoter vectors. The effect of the order of target genes on the gene expression was investigated by comparing the protein productions from the two-promoter vectors that contained VHb-BCL-XL/BAD or BAD/VHb-BCL-XL as the first gene/second gene. In the both vectors, VHb-BCL-XL and BAD were under T5 and T7 promoters, respectively (Figs. 1Go,3Go). We could not detect a noticeable difference in the relative expression levels of the two genes from the comparison set (data not shown), demonstrating that the expression of the two target genes on a two-promoter vector is independent of the order of the genes. Consistently, we observed comparable levels of extremely high expression of CED-4 (first gene) and CED-3 (second gene) from a two-promoter vector, although the two proteins of Caenorhabditis elegans were completely insoluble (data not shown). Similarly, mouse Bet3p and Trs33p, proteins involved in intracellular vesicle transport (Sacher et al. 2000), were produced in similar quantities with a two-promoter vector (data not shown).



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  		Figure 3. Other two-promoter vectors used in this study. Presented are the expression vectors for producing BCL-XL:BAD with the promoter combination or the order of target genes different from that shown in Figure 1AGo.

 
Usually, a highly expressed protein is much easier to purify than a protein expressed at lower level. The superb expression efficiency and successful purification of the six protein complexes in milligram quantity from 1-L culture of E. coli cells demonstrate that the two-promoter system is a significant improvement from the conventional, widely used bicistronic vector system and would serve as the method of the first choice for obtaining large amounts of protein complexes for biochemical and structural studies. We expect that the two-promoter system can be easily extended to a three- or a four-promoter system, and so on, to coexpress more than two proteins, unless a resulting expression vector is excessively large and prevents transformation of E. coli cells.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Construction of bicistronic and two-promoter vectors
The pPosKJ plasmid, containing T5 promoter, was derived from pKW32 by replacing the thrombin cleavage sequence by the TEV cleavage sequence. The pKW32 plasmid is designed to produce a target protein fused to (His)6-tagged VHb (Park et al. 2003). DNA fragments coding for BCL-XL (residues 1–196) and BCL-W (residues 9–152) were amplified from mouse uterus cDNA library (ResGen) and cloned into pPosKJ using the NdeI and NotI restriction sites to produce pPosKJ::BCL-XL and pPosKJ::BCL-W, respectively. DNA fragments coding for BIM-S and BAD were amplified from the same library and ligated into the NdeI and XhoI restriction sites of the pET30a plasmid (Novagen). From these vectors, the DNA fragment containing the RBS-BAD or the RBS-BIM-S sequence was amplified and inserted into pPosKJ::BCL-XL or pPosKJ::BCL-W, to produce the bicistronic vectors. Two-promoter vectors were prepared in the same way except that the inserted fragments contained T7 promoter in front of the RBS-BAD and RBS-BIM-S sequences. The DNA fragments coding for CED-9 (residues 68–251) and EGL-1 were amplified from the C. elegans RB1 cDNA library and genomic DNA, as described (Woo et al. 2003). After introducing the triple mutations of C107S/ C135A/C164S into the CED-9 gene, which was necessary for the solubility of the resulting protein (Woo et al. 2003), the bicistronic and the two-promoter expression vectors for producing the CED-9:EGL-1 complex were created in the same way as described above. The vector containing T7 promoters for both BCL-XL and BAD was generated from pET30a::BCL-XL and pET30a::BAD. To generate the vector containing Trc and T7 promoters, the BCL-XL DNA fragment was first cloned into the pPROEX HTa plasmid (Invitrogen) containing Trc promoter, and then the appropriate DNA fragment from pET30a::BAD was inserted into the resulting pPROEX HTa::BCL-XL. The two-promoter vector containing BAD/VHb-BCL-XL as the first gene/second gene was generated from pET30a::BAD and pPosKJ::BCL-XL.

Expression of protein complexes
The CED-9:EGL-1 complex was expressed using the E. coli BL21 (DE3) pLysS strain. The BCL-XL:BAD, BCL-XL:BIM-S, BCL-XL:BMF, and BCL-W:BAD complexes were expressed using the E. coli BL21 (DE3) RIG strain. An overnight culture of 20 mL was inoculated into 1 L of terrific broth medium, and the cells were grown at 37°C. When the culture reached a log phase (OD600 = 0.6), the expression of the target genes was induced with 1 mM isopropyl {beta}-D-thiogalactoside. The cells were grown for an additional 24 h at 22°C.

Purification of protein complexes
Red-colored, harvested cells from each culture were resuspended in buffer A (20 mM Tris-HCl (pH 8.0) and 5 mM {beta}-mercaptoethanol) and lysed by sonication. The supernatant of the cell lysate was loaded on to a Ni-NTA column (QIAGEN), which was equilibrated with buffer A. After washing the column with buffer A containing 20 mM imidazole, bound proteins were eluted with buffer A containing 300 mM imidazole. Eluted fraction was loaded onto a Hitrap Q anion exchange column (Amersham Biosciences) and the bound proteins were eluted by a 0 to 500 mM linear NaCl gradient. This step removed contaminating proteins further and, in particular, separated the protein complexes from excessive uncomplexed VHb-BCL-XL or VHb-CED-9. The (His)6-tagged VHb protein was cleaved by TEV protease (Invitrogen) with the molar ratio of TEV : protein in 1 : 50 and removed by passing the reaction mixture through a Ni-NTA column.

CD spectroscopy
CD spectra were recorded on a JASCO J-715 spectropolarimeter with protein samples (2.5–10 µM) in 20 mM phosphate buffer (pH 7.5) at 25°C over the range of 200–250 nm in a nitrogen atmosphere. Each spectrum is the accumulation of three scans corrected by subtracting signals from the buffer control.


   Acknowledgments
 
This work was supported by Creative Research Initiatives of the Korean Ministry of Science & Technology.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


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 Introduction
 Results and Discussion
 Materials and methods
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