PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions

Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.     

In planta protein-protein interactions assessed using a nanovirus-based replication and expression system

The multipartite genome of the nanovirus Faba bean necrotic yellows virus, which consists of one gene on each DNA component, was exploited to construct a series of virus-based episomal vectors designed for transient replication and gene expression in plants. This nanovirus based expression system yields high levels of protein which allows isolation of recombinant protein and protein complexes from plant tissues. As examples, we demonstrated in planta interaction between the nanovirus F-box protein Clink and SKP1, a constituent of the ubiquitin-dependent protein turnover pathway. Thus, replicative nanovirus vectors provide a simple and efficient means for in planta characterization of protein-protein interaction.     

Tandem affinity purification vectors for use in gram positive bacteria

Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 degrees C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3xFLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.     

Two versatile shuttle vectors for Thermus thermophilus-Escherichia coli containing multiple cloning sites, lacZα gene and kanamycin or hygromycin resistance marker

Two versatile shuttle vectors for Thermus thermophilus and Escherichia coli were developed on the basis of the T. thermophilus cryptic plasmid pTT8 and E. coli vector pUC13. These shuttle vectors, pTRK1T and pTRH1T, carry a gene encoding a protein homologous to replication protein derived from pTT8, a replicon for E. coli, new multiple cloning sites and a lacZα gene from E. coli vector pUC13, and also have a gene encoding a thermostable protein that confers resistance to kanamycin or hygromycin, which can be used as a selection marker in T. thermophilus. These shuttle vectors are useful to develop enzymes and proteins of biotechnological interest. We also constructed a plasmid, pUC13T, which carries the same multiple cloning sites of pTRK1T and pTRH1T. These vectors should facilitate cloning procedures both in E. coli and T. thermophilus.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Glycoprotein exchange vectors based on vesicular stomatitis virus allow effective boosting and generation of neutralizing antibodies to a primary isolate of human immunodeficiency virus type 1

Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with the G protein gene replaced with G genes from two other VSV serotypes, New Jersey and Chandipura. These G protein exchange vectors grew to titers equivalent to wild-type VSV and induced similar neutralizing titers to themselves but no cross-neutralizing antibodies to the other two serotypes. The effectiveness of these recombinant VSV vectors was illustrated in experiments in which sequential boosting of mice with the three vectors, all encoding the same primary human immunodeficiency virus (HIV) envelope protein, gave a fourfold increase in antibody titer to an oligomeric HIV envelope compared with the response in animals receiving the same vector three times. In addition, only the animals boosted with the exchange vectors produced antibodies neutralizing the autologous HIV primary isolate. These VSV envelope exchange vectors have potential as vaccines in immunizations when boosting of immune responses may be essential.     

Improved bicistronic mammalian expression vectors using expression augmenting sequence element (EASE)

We have recently reported on the isolation of a 5.7 kb segment of Chinese hamster ovary cell genomic DNA, Expression Augmenting Sequence Element (EASE), which when used in bicistronic expression vectors allows the development of stable Chinese hamster ovary cell pools in a five to seven week time period that express high levels of recombinant protein (6–25 μg 10-6 cells/day depending on the protein). In the present study, we have mapped the activity of the EASE to a 2.1 kb region using colony forming assays and developed bicistronic expression vectors with the smaller EASE or control lambda DNA. The recovery of pools expressing the hematopoietic growth factor, FLT3 Ligand, in methotrexate-containing media took 1 to 4 weeks less when using EASE expression vectors compared with control vectors. The cell pools developed with the EASE and control vectors had similar final protein expression levels. Southern blot analysis suggested the expression cassette from the EASE containing vectors integrated in tandem arrays arranged in either head to head or head to tail fashion. By contrast, control vectors appeared to integrate with multiple interruptions to the expression vector. Thus, the EASE, within a bicistronic expression vector, appeared to facilitate tandem vector integration and reduce the time required to develop cell pools for protein expression. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors.

Two improved plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusions, have been constructed. These vectors allow fusion of any gene to the protein A moiety, giving fusion proteins which can be purified, in a one-step procedure by IgG affinity chromatography. One vector, pRIT2, is designed for temperature-inducible expression of intracellular fusion proteins in Escherichia coli and the other pRIT5, is a shuttle vector designed for secretion. The latter gives a periplasmatic fusion protein in E. coli and an extracellular protein in Gram-positive hosts such as Staphylococcus aureus. The usefulness of these vectors is exemplified by fusion of the protein A gene and the E. coli genes encoding the enzymes beta-galactosidase and alkaline phosphatase. High amounts of intact fusion protein are produced which can be immobilized on IgG-Sepharose in high yield (95-100%) without loss of enzymatic activity. Efficient secretion in both E. coli and S. aureus, was obtained for the alkaline phosphatase hybrid, in contrast to beta-galactosidase which was only expressed efficiently using the intracellular system. More than 80% of the protein A alkaline-phosphatase hybrid protein can be eluted from IgG affinity columns without loss of enzymatic activity.     

Selection of novel vesicular stomatitis virus glycoprotein variants from a peptide insertion library for enhanced purification of retroviral and lentiviral vectors

The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering.     

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.     

New vectors for peptide display on the surface of filamentous bacteriophage

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.     

Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.

The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.     

High level expression of genes cloned in phage lambda gt11

Plasmid cloning vectors have been constructed which allow genes originally cloned in lambda gt11 to be expressed at a high level in Escherichia coli. They are based on the pEMBL and pUC vectors, with the genes transcribed from the lac promoter. The EcoRI site in the vector has been altered to be in the same reading frame as the site used for cloning in lambda gt11. Cloned proteins are expressed fused to a 2-kDa leader sequence containing a run of six Aparagine residues which considerably improves the stability of the recombinant proteins, but does not interfere with immunological assays. Using these vectors, the Mycobacterium leprae 18-kDa protein was expressed at 20 mg per litre of culture and constituted 15% of total cell protein.     

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.     

Development of chloroplast transformation vectors, and a new target region in the tobacco plastid genome

Plastid transformation vectors are used for high-level expression of industrially important recombinant proteins in plants. In the present study, new vectors for plastid transformation were developed. One of these vectors targets transgenes at a new site in the chloroplast genome. Intergenic regions of trnfM-trnG, ndhB-trnL and rrn16-trnV were selected as sites for transgene insertion. Tobacco chloroplast was successfully transformed with designed vectors, and the transplastomic plants accumulated recombinant protein as high as 5–6% of total soluble protein which remained localized in the chloroplasts. Although the vectors were designed using the plastid genome of Nicotiana tabacum, flanking regions used in two vectors show a high level of homology with chloroplast genomes of other plant species, thus it might be possible to use them for the transformation of a wider range of plant species.     

A Vector Design that Allows Fast and Convenient Production of Differently Tagged Proteins

Recombinant-tagged proteins have a widespread use in experimental research as well as in clinical diagnostic and therapeutic approaches. Well-stocked sets of differently tagged variants of a same protein would be of great help. However, the construction of differently tagging vectors is a demanding task since cloning procedures need several tailored DNA inserts. In this study, we describe a novel vector system that allows a cost- and time-effective production of differently tagged variants of a same protein by using the same DNA fragment and a set of vectors each carrying a different tag. The design of these expression vectors is based on an intronic region that becomes functional upon cloning the insert sequence, splicing of which attaches a certain tag to the protein termini. This strategy allows for the cloning of the fragment that codes for the protein of interest, without any further modification, into different vectors, previously built and ready-to-use, each carrying a tag that will be joined to the protein. Proof of principle for our expression system, presented here, is shown through the production of a functional anti-GD2 Fab fragment tagged with biotin or polyhistidine, or a combination of both, followed by the demonstration of the functional competencies of both the protein and the tags.     

Ligand-induced conformational change of a protein reproduced by a linear combination of displacement vectors obtained from normal mode analysis

The conformational change of a protein upon ligand binding was examined by normal mode analysis (NMA) based on an elastic-network model (ENM) for a full-atom system using dihedral angles as independent variables. Specifically, we investigated the extent to which conformational change vectors of atoms from an apo form to a holo form of a protein can be represented by a linear combination of the displacement vectors of atoms in the apo form calculated for the lowest-frequency m normal modes (m=1, 2,…, 20). In this analysis, the latter vectors were best fitted to the former ones by the least-squares method. Twenty-two paired proteins in the holo and apo forms, including three dimer pairs, were examined. The results showed that, in most cases, the conformational change vectors were reproduced well by a linear combination of the displacement vectors of a small number of low-frequency normal modes. The conformational change around an active site was reproduced as well as the entire conformational change, except for some proteins that only undergo significant conformational changes around active sites. The weighting factors for 20 normal modes optimized by the least-squares fitting characterize the conformational changes upon ligand binding for these proteins. The conformational changes sampled around the apo form of a protein by the linear combination of the displacement vectors obtained by ENM-based NMA may help solve the flexible-docking problem of a protein with another molecule because the results presented herein suggest that they have a relatively high probability of being involved in an actual conformational change.     

A comprehensive whole genome bacterial phylogeny using correlated peptide motifs defined in a high dimensional vector space

As whole genome sequences continue to expand in number and complexity, effective methods for comparing and categorizing both genes and species represented within extremely large datasets are required. Methods introduced to date have generally utilized incomplete and likely insufficient subsets of the available data. We have developed an accurate and efficient method for producing robust gene and species phylogenies using very large whole genome protein datasets. This method relies on multidimensional protein vector definitions supplied by the singular value decomposition (SVD) of a large sparse data matrix in which each protein is uniquely represented as a vector of overlapping tetrapeptide frequencies. Quantitative pairwise estimates of species similarity were obtained by summing the protein vectors to form species vectors, then determining the cosines of the angles between species vectors. Evolutionary trees produced using this method confirmed many accepted prokaryotic relationships. However, several unconventional relationships were also noted. In addition, we demonstrate that many of the SVD-derived right basis vectors represent particular conserved protein families, while many of the corresponding left basis vectors describe conserved motifs within these families as sets of correlated peptides (copeps). This analysis represents the most detailed simultaneous comparison of prokaryotic genes and species available to date.