Carboxyl terminus of hVIP/mov34 is critical for HIV-1-Vpr interaction and glucocorticoid-mediated signaling

Human immunodeficiency virus, type 1 (HIV-1) vpr is a highly conserved gene among lentiviruses. The diverse functions of Vpr support interactions of this HIV accessory protein with host cell partners of important pathways. hVIP/mov34 (human Vpr Interacting Protein) is one of these identified Vpr ligands. hVIP is a 34-kDa member of the eIF3 family that is vital for early embryonic development in transgenic mice and important in cell cycle regulation. Its interaction with Vpr, however, is not yet clearly defined. Therefore, we constructed a panel of deletion mutants of this cytoplasmic cellular ligand to map the protein domain that mediates its interaction with Vpr. We observed that the carboxyl-terminal region of hVIP is critical for its interaction with Vpr. In the absence of Vpr or HIV infection, full-length hVIP is expressed in the cytoplasm. The cytoplasmic localization pattern of full-length hVIP protein, however, is shifted to a clear nuclear localization pattern in cells expressing both hVIP and Vpr. In contrast, Vpr did not alter the localization pattern of hVIP mutants, which have their carboxyl-terminal domain deleted. The movement of hVIP supported prior work that suggested that Vpr triggers activation of the GR receptor complex. In fact, we also observed that dexamethasone moves hVIP into the nucleus and that glucocorticoid antagonists inhibit this effect. Interestingly, the expression of an hVIP carboxyl-terminal mutant, which is not responsive to Vpr, is also not responsive to dexamethasone. These data illustrate that the carboxyl-terminal domain of hVIP is critical for mediating hVIP-Vpr interaction as well as for its glucocorticoid response. These results support the view that hVIP is a member of the complex array of nucleocytoplasmic shuttling proteins that are regulated by HIV infection and glucocorticoids.     

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.     

A modular set of prokaryotic and eukaryotic expression vectors

A modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. Special features of these vectors include (i) serial affinity tags (hexahistidine-GST) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (TEV) protease, (iii) PCR cloning design that results in a product of proteolytic cleavage with only one (a single glycine) or two (gly-ala) amino acids at the N-terminus of the protein, and (iv) expression in either Escherichia coli or Saccharomyces cerevisiae. In addition, singly hexahistidine-tagged proteins can be produced for purification under denaturing conditions and some vectors allow addition of five amino acid kinase recognition sites for easy radiolabeling of proteins. To illustrate the use of these vectors, all regulatory components of the yeast GAL regulon, rather than abundant highly soluble proteins, were produced and purified under native or denaturing conditions, and their biological activity was confirmed.     

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.     

Gateway-compatible vectors for plant functional genomics and proteomics

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.     

pAUL: A Gateway-Based Vector System for Adaptive Expression and Flexible Tagging of Proteins in Arabidopsis

Determination of protein function requires tools that allow its detection and/or purification. As generation of specific antibodies often is laborious and insufficient, protein tagging using epitopes that are recognized by commercially available antibodies and matrices appears more promising. Also, proper spatial and temporal expression of tagged proteins is required to prevent falsification of results. We developed a new series of binary Gateway cloning vectors named pAUL1-20 for C- and N-terminal in-frame fusion of proteins to four different tags: a single (i) HA epitope and (ii) Strep-tagIII, (iii) both epitopes combined to a double tag, and (iv) a triple tag consisting of the double tag extended by a Protein A tag possessing a 3C protease cleavage site. Expression can be driven by either the 35 S CaMV promoter or, for C-terminal fusions, promoters from genes encoding the chloroplast biogenesis factors HCF107, HCF136, or HCF173. Fusions of the four promoters to the GUS gene showed that endogenous promoter sequences are functional and drive expression more moderately and consistently throughout different transgenic lines when compared to the 35 S CaMV promoter. By testing complementation of mutations affected in chloroplast biogenesis factors HCF107 and HCF208, we found that the effect of different promoters and tags on protein function strongly depends on the protein itself. Single-step and tandem affinity purification of HCF208 via different tags confirmed the integrity of the cloned tags.     

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.     

Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli

For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies.     

A set of baculovirus transfer vectors for screening of affinity tags and parallel expression strategies

We report a set of baculovirus transfer vectors for parallel expression of proteins in fusion with a panel of affinity tags including GST, protein A, thioredoxin, CBP, and FLAG. This suite includes vectors to generate recombinant baculovirus by homologous recombination in insect cells or using the Bac-to-Bac technology. An application of the vector suite approach to the vitamin D receptor (VDR), a protein mainly expressed as inclusion bodies in Escherichia coli, is presented. We found that expression in fusion with GST and protein A provided an efficient compromise of excellent purification with acceptable yields and costs.     

Protein expression in Drosophila Schneider cells

We expressed recombinant secreted, membrane, and cytosolic proteins in stably transfected Drosophila Schneider (SL-3) cells. To allow easy cloning of N- and C-terminal fusion proteins containing epitope- and His-tags for the detection of recombinant proteins and purification by affinity chromatography we constructed new expression vectors. To exemplify the general applicability of protein expression in Schneider cells we characterized the expression system with respect to inducibility, localization of the recombinant proteins, yields of purified proteins, and presence of posttranslational and cotranslational modifications. Secreted proteins became quantitatively N-glycosylated in SL-3 cells and the N-glycan of a Golgi-resident membrane protein was found to be Endo-H-resistant. Myristoylation of AnxXIIIb, a member of the annexin family, could be demonstrated and glycosylphosphatidylinositol-anchored proteins containing their lipid anchor were expressed efficiently in SL-3 cells. Since generation of stable cell lines and mass culture of SL-3 cells is cheap and easy, they provide an attractive eukaryotic expression system.     

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.     

High level expression and single-step purification of hexahistidine-tagged L-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set

Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged L-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV.     

戊型肝炎病毒ORF1多聚蛋白的不同功能域在细胞内的定位

为了探讨戊型肝炎病毒多聚蛋白ORF1的多个功能域在宿主细胞中的表达和定位情况,我们首先将psk-HEV重组载体上的ORF1各功能域的编码序列克隆到绿色荧光蛋白载体pcDNA3.1-GFP上,构建成融合表达的重组质粒,并测序和酶切鉴定其构建成功。再通过Western-Blot验证各融合蛋白在细胞中正确表达,并用激光扫描共聚焦显微镜观察融合蛋白在细胞内的分布和定位。在Huh7细胞中,RdRp蛋白主要分布于细胞核内,HEL蛋白以囊泡状分布于细胞核周,MET蛋白以颗粒状存在于细胞核和细胞质中,PLP蛋白呈极性分布于细胞核周,X蛋白在细胞核和细胞质中均存在。各融合蛋白在细胞中的不同定位印证了对这些蛋白质的功能预测和体外研究结果,这为进一步研究HEV不同蛋白功能提供了支持。     

High level expression and single-step purification of hexahistidine-tagged L-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set

Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins. In many cases, there may be the necessity to remove the tag after protein preparation to regain activity. Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease. Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively. The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors. Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification. Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging. The expression and purification of His-tagged L-2-hydroxyisocaproate dehydrogenase yields fully active enzyme. The tag removal is here accomplished by a derivative of rTEV.     

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.     

新型冠状病毒RBD蛋白原核表达及多克隆抗体的制备

为原核表达严重急性呼吸综合征冠状病毒2（简称新型冠状病毒,severe acute respiratory syndrome-coronavirus 2,SARS-CoV-2）S蛋白受体结合域（receptor binding domain, RBD）并制备多克隆抗体,利用基因克隆技术将RBD基因连接到原核表达载体pGEX-6p-1和pET-32a(+)上,电转化至大肠杆菌XL1-Blue感受态细胞,利用优化后的表达条件大量表达重组蛋白,经亲和层析纯化后通过SDS-PAGE检测蛋白的表达情况。利用GST-RBD融合蛋白作为免疫抗原免疫小鼠制备多克隆抗体,ELISA和Western blot分析抗血清的效价和特异性。PCR鉴定和序列测定结果显示,成功构建了重组载体pGEX-RBD和pET-RBD,在大肠杆菌中实现了GST-RBD和RBD-His融合蛋白的可溶性高效表达。研究获得的多克隆抗体的滴度达到约1∶3 000,并具有良好的结合特异性。原核表达的可溶性新型冠状病毒RBD重组蛋白具有良好的免疫原性,为后续制备基因工程抗体奠定了实验基础。