A new vector for high-throughput,ligation-independent cloning encoding a tobacco etch virus protease cleavage site

To establish high-throughput methods for protein crystallography, all aspects of the production and analysis of protein crystals must be accelerated. Automated, plate-based methods for cloning, expression, and evaluation of target proteins will help researchers investigate the vast numbers of proteins now available from sequenced genomes. Ligation-independent cloning (LIC) is well suited to robotic cloning and expression, but few LIC vectors are available commercially. We have developed a new LIC vector, pMCSG7, that incorporates the tobacco etch virus (TEV) protease cleavage site into the leader sequence. This protease is highly specific and functions under a wide range of conditions. The new vector incorporates an N-terminal his-tag followed by the TEV protease recognition site and a SspI restriction site used for LIC. The vector functioned as expected, giving high cloning efficiencies and strong expression of proteins. Purification and cleavage of a target protein showed that the his-tag and the TEV cleavage site function properly. The protein was purified and cleaved under different conditions to simulate both plate-based screening methods and large-scale purifications for crystal production. The vector also includes a pair of adjacent, unique restriction sites that will allow insertion of additional modules between the his-tag and the cleavage site of the leader sequence to generate a family of vectors suitable for high-throughput production of proteins.     

New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols

The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, ligation-independent cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8–15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.     

A Pair of Ligation-Independent Escherichia coli Expression Vectors for Rapid Addition of a Polyhistidine Affinity Tag to the N- or C-Termini of Recombinant Proteins

6×His tag is one of the most widely used affinity fusion tags that facilitates detection and purification of recombinant proteins. However, the location of this tag within a particular type of protein may influence the expression, solubility, and bioactivity of the protein, and the optimal location needs to be determined experimentally. To provide a tool for rapid generation of 6× His tags at the N- or C-terminus of any recombinant protein, we have constructed a pair of Escherichia coli expression vectors—pLIC-NHis and pLIC-CHis—based on the pET30a vector, for ligation-independent cloning (LIC). Construction of this new pair of LIC vectors was accomplished by replacement of the multiple cloning site of pET30a with two specifically designed LIC cloning sites. A target gene derived by PCR with a pair of predesigned primers can be inserted into the LIC site of pLIC-NHis for expression of recombinant proteins fused with the N-terminal sequence MHHHHHHG or into that of pLIC-CHis for expression of recombinant proteins with the C-terminal sequence THHHHHH. Successful expression of two normal mammalian prion proteins and five bacterial proteins in E. coli using this pair of LIC vectors reveals that these vectors are valuable tools for the production of recombinant His-tagged proteins in E. coli.     

Positive-selection and ligation-independent cloning vectors for large scale <Emphasis Type="Italic">in Planta</Emphasis> expression for plant functional genomics

Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3′ to 5′ exonuclease activity in the presence of only one dNTP (dGTP for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants.     

Ligation independent cloning vectors for expression of SUMO fusions

With demand increasing for the production of many different proteins for biophysical or biochemical analyses, rapid methods are needed for the cloning, expression and purification of native recombinant proteins. In particular, generic methods are required that are independent of the target gene sequence. To address this challenge we have constructed four Escherichia coli expression vectors that can be used for ligation independent cloning (LIC) of an amplified target gene sequence. These vectors represent the combinatorial pairing of two different parent vector backbones with two different affinity tags. The target gene is cloned downstream of the sequence coding for an affinity-tagged small ubiquitin related modifier (SUMO). Using enhanced green fluorescent protein (eGFP) as an example we demonstrate that the LIC procedure works with high efficiency for all four of the vectors. We also show that the resultant recombinant SUMO fusion proteins can be overexpressed in E. coli and readily isolated by standard affinity purification techniques. Importantly, the purified fusion product can be treated with recombinant SUMO hydrolase to yield a mature target protein with any residue except proline at the amino terminus. We demonstrate an application of this by generating recombinant eGFP containing a non-native amino terminal cysteine residue and using it as a substrate for expressed protein ligation (EPL). The reagents and techniques described here represent a generic method for the rapid cloning and production of a target protein, and would be appropriate for a high throughput genomic scale expression project.     

New LIC vectors for production of proteins from genes containing rare codons

In the effort to produce proteins coded by diverse genomes, structural genomics projects often must express genes containing codons that are rare in the production strain. To address this problem, genes expressing tRNAs corresponding to those codons are typically coexpressed from a second plasmid in the host strain, or from genes incorporated into production plasmids. Here we describe the modification of a series of LIC pMCSG vectors currently used in the high-throughput (HTP) production of proteins to include crucial tRNA genes covering rare codons for Arg (AGG/AGA) and Ile (AUA). We also present variants of these new vectors that allow analysis of ligand binding or co-expression of multiple proteins introduced through two independent LIC steps. Additionally, to accommodate the cloning of multiple large proteins, the size of the plasmids was reduced by approximately one kilobase through the removal of non-essential DNA from the base vector. Production of proteins from core vectors of this series validated the desired enhanced capabilities: higher yields of proteins expressed from genes with rare codons occurred in most cases, biotinylated derivatives enabled detailed automated ligand binding analysis, and multiple proteins introduced by dual LIC cloning were expressed successfully and in near balanced stoichiometry, allowing tandem purification of interacting proteins.     

In vivo biotinylated proteins as targets for phage-display selection experiments

Screening phage-displayed combinatorial libraries represents an attractive method for identifying affinity reagents to target proteins. Two critical components of a successful selection experiment are having a pure target protein and its immobilization in a native conformation. To achieve both of these requirements in a single step, we have devised cytoplasmic expression vectors for expression of proteins that are tagged at the amino- or carboxy-terminus (pMCSG16 and 15) via the AviTag, which is biotinylated in vivo with concurrent expression of the BirA biotin ligase. To facilitate implementation in high-throughput applications, the engineered vectors, pMCSG15 and pMCSG16, also contain a ligase-independent cloning site (LIC), which permits up to 100% cloning efficiency. The expressed protein can be purified from bacterial cell lysates with immobilized metal affinity chromatography or streptavidin-coated magnetic beads, and the beads used directly to select phage from combinatorial libraries. From selections using the N-terminally biotinylated version of one target protein, a peptide ligand (Kd= 9 microM) was recovered that bound in a format-dependent manner. To demonstrate the utility of pMCSG16, a set of 192 open reading frames were cloned, and protein was expressed and immobilized for use in high-throughput selections of phage-display libraries.     

大肠杆菌多基因共表达策略

利用基因工程手段实现多个基因在同一宿主菌中共表达是大肠杆菌细胞发育调节研究和代谢途径改造的有效手段。介绍了单一转录单元的多基因共表达载体、多重转录单元的多基因共表达和单基因载体的构建原理、特点、优势及转化策略,并着重介绍了利用LIC衔接子实现基因在多基因载体上定位连接的原理和方法。     

Vectors for co-expression of an unrestricted number of proteins

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells.     

Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5′ LIC site of expression constructs and report on the construction of expression vectors with N-terminal serine, cysteine, threonine, or tyrosine residues after tobacco etch virus (TEV) protease cleavage. In a practical application, the N-terminal serine was oxidized to an aldehyde, subsequently reacted with an amino-oxy functionalized polyethylene glycol (PEG) ligand under aniline catalysis to provide a protein selectively modified at the N-terminus.     

Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags

Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1beta in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.     

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.     

InsectDirectTM System: Rapid, High-level Protein Expression and Purification from Insect Cells

A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli, insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells (Sf9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirect^TM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases.     

Enzyme Free Cloning for high throughput gene cloning and expression

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning (EFC) procedure, a PCR-only method that eliminates all variables other than PCR efficiency by circumventing enzymatic treatments. We compared the cloning efficiency of EFC with that of Ligation Independent Cloning (LIC). Both methods are well suited for HTP cloning, but EFC yields three times more transformants and a cloning efficiency of 91%, comparable with recombinational cloning methods and significantly better than LIC (79%). EFC requires only nanogram amounts of both vector and insert, does not require highly competent cells and is, in contrast to LIC, largely insensitive to variations in PCR product concentration. Automated protein expression screening of expression strains directly transformed with EFC reactions showed, that the traditional preceding step via a cloning strain can be circumvented. EFC proves an efficient and robust HTP cloning method, that is compatible with existing Ligation Independent Cloning vectors, and highly suitable for automation.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Simultaneous cloning of open reading frames into several different expression vectors

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.     

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.