Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum

We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.     

Evidence for the role of glycosylation in accessibility of the extracellular domains of human MRP1 (ABCC1)

To enable cell surface localization of the human multidrug resistance protein (MRP1, ABCC1) and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of the three membrane-spanning domains (MSDs) of the transporter. We constructed and expressed various partially and fully glycosylation-deficient, FLAG-tagged MRP1 proteins in a Vaccinia virus-based transient expression system, and the cell surface expression level of MRP1 on intact cells was followed by flow cytometry, using the FLAG tag specific monoclonal antibody M2. We also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in stably transfected HEK293 cells, and followed the cell surface expression and the transport function of MRP1 both by monitoring the efflux of fluorescent substrate and by their ability to confer resistance to HEK293 transfectants to anticancer agents such as daunorubicin and etoposide. When we inserted the FLAG epitope in extracellular loops of the MSD1 or MSD3, the tag was accessible upon removal of N-glycosylation sites (N --> Q at positions 17, 23, and 1006, respectively), whereas the FLAG epitope placed in the MSD2 was not accessible even after removal of all three N-glycosylation sites, indicating that MSD2 region is deeply buried in the plasma membrane. However, all FLAG tagged MRP1 mutants were expressed at the cell surface to the same extent as the wild-type protein and also exhibited normal transport function. Our results demonstrate that the accessibility of the external FLAG epitope is strongly dependent on the position of the tag and the glycosylation state of the different FLAG-tagged MRP1s, and the conformation of extracellular loops in MSD1 and MDS3 does not appear to contribute to the functional status of MRP1.     

Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein(+), yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.     

Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein

We have studied the cleavage efficiency of the protease enterokinase (EK) using the novel vector pESP4. pESP4 is a yeast expression vector equipped with ligation-independent cloning sites, a GST purification tag, and a FLAG epitope tag. EK is used to cleave the FLAG and GST tags leaving the protein of interest without any extraneously added amino acids. We have found that EK is relatively permissive of the amino acid residue downstream of the recognition sequence (the P'1 position). This makes EK an ideal choice to use as a protease to cleave any protein of interest cloned within the pESP4 yeast expression vector.     

Membrane topology of the murine fatty acid transport protein 1

The murine fatty acid transport protein (FATP1) was identified in an expression cloning screen for proteins that facilitate transport of fatty acids across the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning domains. To test this model, we inserted a hemagglutinin epitope tag at the amino terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and expressed these constructs in NIH 3T3 cells. Both tagged constructs produce proteins of the expected molecular masses and are functional in fatty acid import assays. Indirect immunofluorescence studies with selective permeabilization conditions and protease protection studies of sealed membrane vesicles from cells expressing epitope-tagged FATP1 were performed. These experiments show that the extreme amino terminus of tagged FATP1 is oriented toward the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs containing predicted membrane-associated or soluble portions of FATP1 were expressed in Cos7 cells and analyzed by immunofluorescence and subcellular fractionation. These experiments demonstrate that amino acids 1-51, 52-100, and 101-190 contain signals for integral association with the membrane, whereas residues 258-313 and 314-475 are only peripherally membrane-associated. Amino acid residues 191-257 and 476-646 do not direct membrane association and likely face the cytosol. Taken together, these data support a model of FATP1 as a polytopic membrane protein with at least one transmembrane and multiple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty acid transport proteins.     

Multiple epitope tagging of expressed proteins for enhanced detection

Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7. The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system. The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells. In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated.     

Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein⁺, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.     

A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.     

Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea)

Chimeric constructs with the hydrophilic octapeptide FLAG epitope (DYKDDDDK) have been widely used as multipurpose tags for identification, detection, and purification of FLAG fusion proteins. Constructs consisting of C-terminal FLAG-tagged genomic and cDNA clones of anArabidopsis phytochelatin synthase gene,AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression ofAtPCS1 in transgenic lines were confirmed by using PCR and Northern blot analyses. However, immunoblot analysis revealed strong nonspecific binding of a monoclonal anti-FLAG M2 antibody to an endogenous protein in both shoot and leaf tissues of wild-type Indian mustard (85-kDa) that masked presence of the phytochelatin synthase (PCS) protein of interest (55-kDa). Further analysis revealed absence of a nonspecific protein in root tissues of transgenic plants, thus allowing detection of the FLAG-tagged PCS protein.     

Mimicking rubella virus particles by using recombinant envelope glycoproteins and liposomes.

The envelope glycoproteins E1 and E2 of rubella virus (RV) were engineered to display the FLAG epitope tag and a polyhistidine tag, at their amino and carboxy termini, respectively. These modified envelope proteins were produced in Sf9 insect cells utilizing baculovirus expression vectors, the E1 and E2 vectors giving rise to protein products of about 58 and 42 kDa, respectively. The recombinant proteins were purified by immobilized metal-ion affinity chromatography and reconstituted into liposomes via their hydrophobic transmembrane anchors. The liposomes were prepared by detergent dialysis in the presence of europium-DTPA chelate, enabling the subsequent measurement of the binding of the resultant proteoliposomes to the antibodies by time resolved fluorescence. RV mimicking proteoliposomes were recognized by antibodies specific for the E1 and E2 proteins, as well as the FLAG epitope tag. This type of virosome may prove useful for studies on the basic biological events of an RV infection or as diagnostic reagents.     

利用人U6 snRNA启动子构建RNA干扰质粒载体

RNA干扰技术已经成为基因功能研究等领域的有力工具,构建带有筛选标记的siRNA载体可以在细胞中持续抑制靶基因的表达.为了利用RNAi技术开展生物学研究,在克隆载体pUC19的基础上改造构建了人类细胞小干扰RNA（small interference RNA,siRNA）表达质粒pUC19NU.该质粒具有新霉素抗性标记和真核细胞复制起点,利用连入的人U6 snRNA启动子起始siRNA的转录.以EGFP 和p53为靶基因的干扰实验证明,所构建的siRNA表达质粒可以显著抑制细胞外源性增强绿色荧光蛋白（enhanced green fluorescent protein,EGFP）及细胞内源性p53蛋白的表达,而且抑制效果具有特异性.     

Functional Characterization and Potential Applications for Enhanced Green Fluorescent Protein- and Epitope-Fused Human M1 Muscarinic Receptors

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.     

Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.     

Overexpression of a heterologous protein,haloalkane dehalogenase,in a poly-beta-hydroxybutyrate-deficient strain of the facultative methylotroph Methylobacterium extorquens AM1

Using an expression vector containing p(mxaF'), a strong native promoter, expression of a model heterologous protein, haloalkane dehalogenase, from Xanthobacter autotrophicus GJ10 was achieved in the methylotrophic bacterium, Methylobacterium extorquens AM1. Although expression using the wild-type strain was <5% of total cell protein, expression at a level of 10% of the total cell protein was achieved in a mutant unable to synthesize poly-beta-hydroxybutyrate granules. Two other tested heterologous proteins, catechol dioxygenase and green fluorescent protein, were expressed at moderate levels in both wild-type and the PHB-negative strain. These results suggest that the M. extorquens PHB-negative strain is a possible platform for overexpression of heterologous proteins with labeled or unlabeled methanol as a starting material.     

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3?×?FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3?×?FLAG tag, Twin-Strep tag, or CBP-3?×?FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.     

Engineered Mammalian Vector to Express EGFP-Tagged Proteins as Biomarkers

Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvβ3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvβ3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.     

Minimal FLAG sequence useful in the functional epitope tagging of H-Ras

Epitope tagging can interfere with normal protein function, indicating the need for an unobtrusive epitope tag. The FLAG epitope (DYKDDDDK) was examined for a minimal epitope useful in the tagging of H-Ras. The heptapeptide tag, F7 (MDYKDDD), was found to retain reactivity with M2 and M5 monoclonal antibodies in immunoprecipitation, Western blotting, and immunofluorescence microscopy. The F7 tag did not interfere with Ras stability, EGF stimulation of Ras activation, and downstream phosphorylation of MAPK Erk1/2. Unlike the full FLAG sequence, the F7 tag had minimal effect on the growth properties of H-Ras in a colony-forming assay. The F7 tag may be useful when minimizing the effect of tagging on protein function is an important criterion in the selection of an N-terminal epitope tag.     

Visualization of proteins in intact cells with a clonable tag for electron microscopy

Identification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.     

集胞藻PCC 6803中脂肪酸激活酶Slr1609互作蛋白的鉴定

集胞藻中slr1609是编码脂肪酸激活酶的基因,对与其相关的重要功能伴侣蛋白进行研究,可以完善对脂肪酸合成模块的认识,为进一步通过合成生物学技术改造蓝细菌提供理论支持。本研究在集胞藻PCC 6803中建立了蛋白质复合体分析及鉴定技术:利用氯霉素抗性基因筛选,构建带有3×FLAG标签的Slr1609突变株,通过RT-PCR优化重组蛋白表达条件;同时对突变株进行了Western blotting鉴定,以及利用Native-PAGE验证了蛋白质复合体的存在。最后,LC-MS/MS质谱鉴定获得了Slr1609蛋白复合体中的可能伴侣蛋白。