New Shuttle Vector-Based Expression System To Generate Polyhistidine-Tagged Fusion Proteins in Staphylococcus aureus and Escherichia coli.

Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins. Vectors pBUS1-HC and pTSSCm have no promoter upstream of the multiple cloning site (MCS), and this allows study of genes under the control of their native promoters, and pBUS1-Pcap-HC and pTSSCm-Pcap contain the strong constitutive promoter of S. aureus type 1 capsule gene 1A (Pcap) upstream of a novel MCS harboring codons for the peptide tag Arg-Gly-Ser-hexa-His (rgs-his6). All plasmids contained the backbone derived from pBUS1, including the E. coli origin ColE1, five copies of terminator rrnB T1, and tetracycline resistance marker tet(L) for S. aureus and E. coli. The minimum pAMα1 replicon from pBUS1 was improved through either complementation with the single-strand origin oriL from pUB110 (pBUS1-HC and pBUS1-Pcap-HC) or substitution with a pT181-family replicon (pTSSCm and pTSSCm-Pcap). The new constructs displayed increased plasmid yield and segregational stability in S. aureus. Furthermore, pBUS1-Pcap-HC and pTSSCm-Pcap offer the potential to generate C-terminal RGS-His6 translational fusions of cloned genes using simple molecular manipulation. BcgI-induced DNA excision followed by religation converts the TGA stop codon of the MCS into a TGC codon and links the rgs-his6 codons to the 3' end of the target gene. The generation of the rgs-his6 codon-fusion, gene expression, and protein purification were demonstrated in both S. aureus and E. coli using the macrolide-lincosamide-streptogramin B resistance gene erm(44) inserted downstream of Pcap. The new His tag expression system represents a helpful tool for the direct analysis of target gene function in staphylococcal cells.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot

Latex glycoprotein (LGP) from Synadenium grantii latex was purified by the combination of heat precipitation and gel permeation chromatography. LGP is a heat stable protein even at 80 degrees C showed a sharp single band both in SDS-PAGE as well as in native (acidic) PAGE. LGP is a monomeric protein appears as single band under reducing condition. It is a less hydrophobic protein showed sharp single peak in RP-HPLC with retention time of 13.3 m. The relative molecular mass of LGP is 34.4 kDa. CD spectrum of LGP explains less content of alpha-helix (7%), and high content of beta-pleated sheets (48%) and random coils (46%). The N-terminal sequence of LGP is D-F-P-S-D-W-Y-A-Y-E-G-Y-V-I-D-R-P-F-S. Purified LGP is a fibrinogen degrading protease hydrolyses all the three subunits in the order of Aalpha, Bbeta and gamma. The hydrolytic pattern is totally different from plasmin as well as thrombin. LGP reduces recalcification time from 165 to 30 s with citrated human plasma but did not show thrombin like as well as factor Xa-like activity. Although LGP induces procoagulant activity, it hydrolyses partially cross-linked fibrin clot. It hydrolyses all the subunits of partially cross-linked fibrin clot (alpha- chains, beta-chain and gamma-gamma dimer). LGP is a serine protease, inhibited by PMSF. Other serine protease inhibitors, aprotinin and leupeptin did not inhibit the caseinolytic activity as well as fibrinogenolytic activity. We report purification and characterization of a glycoprotein from Synadenium grantii latex with human fibrino(geno)lytic activity.

Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Expression, purification and low-resolution structure of human vitamin C transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

Purification and characterization of a specific antigen from Echinococcus multilocularis.

A polypeptide (Em2a) purified by affinity chromatography from the Echinococcus multilocularis metacestode showed a high degree of purity as assayed by SDS-PAGE and analytical isoelectrical focusing. A minor contamination with host albumin was revealed. Estimation of relative mol. mass gave a value of 54,000. The isoelectric point was found to be 4.8. Antigenic activity of the polypeptide was demonstrated by immunoprecipitation and western blotting. In these assays the protein was recognized only by homologous sera from patients infected with larval E. multilocularis. This antigen (Em2a) did not react in the ELISA with sera from patients infected with heterologous helminths; these sera were highly cross-reacting with antigen from E. granulosus hydatid fluid. Seventy-three (94%) from 78 investigated patients (alveolar echinococcosis) showed a seropositive reaction with the polypeptide Em2a.

Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis.

Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein.

U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.