A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid

ABSTRACT: BACKGROUND: The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. RESULTS: A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. CONCLUSIONS: The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.     

Gateway RFP-fusion vectors for high throughput functional analysis of genes

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.     

Parallel gene cloning and protein production in multiple expression systems

To quickly find an optimal expression system for recombinant protein production, a set of vectors with the same restriction sites were constructed for parallel cloning of a target gene and recombinant protein production in prokaryotic and eukaryotic expression systems, simultaneously. These vectors include nucleotide sequences encoding protein tags and protease recognition sites for tag removal, followed by the cloning sites 5′‐EcoRI/3′‐XhoI identical in these vectors for ligating with the sticky‐end PCR product of a target gene. Our vectors allow parallel gene cloning and protein production in multiple expression systems with minimal cloning effort. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A simple and efficient expression and purification system using two newly constructed vectors

Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility screening and purification efficiency. The system is based on two newly constructed vectors, pEHISTEV and pEHISGFPTEV derived from a pET vector. Both vectors generate a construct with an amino-terminal hexahistidine tag (His-tag). In addition, pEHISGFPTEV expresses a protein with an N-terminal His-tagged green fluorescent protein (GFP) fusion to allow rapid quantitation of soluble protein. Both vectors have a tobacco etch virus (TEV) protease cleavage site that allows for production of protein with only two additional N-terminal residues and have the same multiple cloning site which enables parallel cloning. Protein purification is a simple two-stage nickel affinity chromatography based on the His tag removal. A total of seven genes were tested using this system. Expression was optimised using pEHISGFPTEV constructs by monitoring the GFP fluorescence and the soluble target proteins were quantified using spectrophotometric analysis. All the tested proteins were purified with sufficient quantity and quality to attempt structure determination. This system has been proven to be simple and effective for structural biology. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable.     

Expression vectors for enzyme restriction- and ligation-independent cloning for producing recombinant His-fusion proteins

In this work we have constructed two novel expression vectors, designated as pURI2 and pURI3, which enable parallel cloning of a given target gene for producing recombinant His-fusion proteins. The vectors were created using the well-known pT7-7 and pIN-III-A3 plasmids as their template. The same DNA fragment containing the His-tag, enterokinase cleavage site, and a NotI unique site, as well as keeping the HindIII unique restriction site, was introduced in both vectors. These vectors have been designed to avoid the enzyme restriction and ligation steps during the cloning. The unique NotI site was introduced to facilitate the selection of the adequate recombinant plasmid. Parallel cloning of the same polymerase chain reaction fragment can be carried out since both vectors shared the same leader sequence. The described strategy avoids tedious cloning efforts into different expression vectors and represents a highly efficient means of cloning. To validate our vectors, we have cloned one target gene in both vectors and used expression and purification techniques to obtain the recombinant target protein. We herein show that both vectors function effectively in all the required experimental steps-cloning, expression, purification, and cleavage.     

Differential screening of phage-ab libraries by oligonucleotide microarray technology

A novel and efficient tagArray technology was developed that allows rapid identification of antibodies which bind to receptors with a specific expression profile, in the absence of biological information. This method is based on the cloning of a specific, short nucleotide sequence (tag) in the phagemid coding for each phage-displayed antibody fragment (phage-Ab) present in a library. In order to set up and validate the method we identified about 10,000 different phage-Abs binding to receptors expressed in their native form on the cell surface (10 k Membranome collection) and tagged each individual phage-Ab. The frequency of each phage-Ab in a given population can at this point be inferred by measuring the frequency of its associated tag sequence through standard DNA hybridization methods. Using tiny amounts of biological samples we identified phage-Abs binding to receptors preferentially expressed on primary tumor cells rather than on cells obtained from matched normal tissues. These antibodies inhibited cell proliferation in vitro and tumor development in vivo, thus representing therapeutic lead candidates.     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells.     

Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases

High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.     

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway^® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community.     

p∆TubHA4C, a new versatile vector for constitutive expression in Drosophila

Several vectors for gene expression are available in Drosophila, a hub for genetics and genomics innovation. However, the vectors for ubiquitous expression have a complex structure, including coding exons, that makes in-frame cloning of cDNAs very complicated. In this report we describe a new Drosophila expression vector (p?TubHA4C) for ubiquitous expression of coding sequences under the control of a minimal 0.9 kb promoter of α1 tubulin (α1t). This plasmid was designed to include optimized multiple cloning sites (polylinker) to provide flexibility in cloning strategies. We also added the option of double labeling the expressed proteins with two C-terminal tags, the viral epitope hemagglutinin and a synthetic tetracysteine (4C) tag that binds small fluorescent compounds. This dual tag allows both in situ and biochemical detection of the desired protein. In particular, the new 4C tag technology combines easy fluorescent labeling with small arsenical compounds in live or fixed cells and tissues, while producing minimal alterations to the tagged protein due to its small size. To demonstrate the potent and ubiquitous expression under the control of the ?Tub promoter, bacterial lacZ was expressed and monitored in cell culture and transgenic flies. We found that the modified 0.9 kb ΔTub promoter induced similar expression levels to the intact 2.6 kb α1t promoter, supporting the inclusion of all critical regulatory elements in the new and flexible ?TubHA4C vector.     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway? recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio? fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor

Tagged G‐protein‐coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N‐terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C‐terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N‐terminus, or EGFP located at the C‐terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 56–68, 2002     

An improved vector system for insertional gene inactivation inspired by the tmRNA-tagging system of S. pneumoniae

Insertional mutagenesis is a technique often used to inactivate genes in Streptococcus pneumoniae. Using conventional vectors, a 5' segment of the targeted gene remains under the control of the gene's authentic promoter following gene disruption. Thus, the expression of a functional peptide and the misinterpretation of results in consequence cannot be excluded. To circumvent this problem, we have developed a plasmid for insertional mutagenesis based on the tmRNA-tagging system of S. pneumoniae which ensures that any protein expressed after gene disruption is degraded. Insertional mutagenesis using this vector results in the targeted gene being tagged with a tmRNA-derived sequence coding for a proteolysis tag. Here we show that the translation product of a gene tagged by this method is not detectable by Western blotting, suggesting that the protein was degraded. This modified vector allows total inactivation of genes with a reliability that cannot be achieved by conventional vectors for insertional mutagenesis. This approach can be applied to other bacterial species.     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His₆-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.     

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.