High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.     

Identification, immunoaffinity purification and partial characterization of a human decidua-associated protein

A crude extract of pooled early-pregnancy decidual tissue was enriched for soluble decidual proteins by exhaustive affinity absorption with antibodies to human serum proteins immobilized on Eupergit C. The partly purified extract was used to prepare monoclonal antibodies. A monoclonal antibody was obtained recognizing an antigen present in extract of decidual tissue and not in extract of proliferative endometrium. The monoclonal antibody was used for immunoaffinity purification of the decidua-associated protein. By SDS-PAGE analysis, under reducing conditions it yielded 2 bands at apparent molecular weights of 55,000 and 25,000. Under non-reducing conditions a single protein band at apparent molecular weight of 200,000 was observed. The Mr 200,000 protein was named hDP200 and the Mr 55,000 protein was named hDP55. It is suggested that hDP55 is a subunit of the hDP200. The hDP200 did not react with polyclonal antibodies specific for PP12 and PP14. PP14 has been shown to be immunologically indistinguishable from PEP and alpha 2-PEG. Our data therefore suggest that hDP200 is a novel human decidua-associated protein.     

High-level bacterial expression, purification and characterization of human calreticulin.

To investigate its cellular function and role in autoimmune disease pathogenesis, we have bacterially expressed human calreticulin, a major calcium-binding protein in the endoplasmic reticulum and a human autoantigen. This is the first report describing the heterologous expression of calreticulin from any source. The recombinant calreticulin constituted approximately 32% of the soluble Escherichia coli proteins, and was purified to apparent homogeneity by ion exchange and hydrophobic liquid chromatography. As does the bona fide protein, the recombinant calreticulin binds calcium and undergoes changes in its conformation upon Zn2+ binding. We take this as a strong indication that the folding of the E.coli-expressed calreticulin is very similar, if not identical, to that of the authentic protein. Moreover, the bacterially expressed calreticulin readily reacted with anti-human and anti-rabbit antibodies, and the anti-recombinant calreticulin antibodies immunoreacted with HeLa calreticulin. The availability of this expression system will allow us to carry out site-specific and deletion mutagenesis analysis in structure--function studies of calreticulin.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli

A T7 promoter-based His6-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus. The vector allows overproduction and single-step purification of His6-fusion protein by immobilized metal (Ni2+) chelate affinity chromatography. The gene encoding leucyl-tRNA synthetase (leuS) was cloned into this vector and expressed in high level. The specific activity of the synthetase in the crude extract of E. coli JM109(DE3) transformant containing the His6-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector). The overproduced His6-fusion leucyl-tRNA synthetase can be purified to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55%. The His6-tag at the N terminus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Aquifex aeolicus

Aquifex aeolicus leucyl-tRNA synthetase is the only known heterodimeric LeuRS, consisting of two subunits with molecular masses of 74.0 and 33.5 kDa, and named alphabeta-LeuRS. The gene encoding alpha subunit was cloned into pSBET-b vector. Synthetic oligonucleotide encoding six histidine residues was also inserted in front of alpha subunit. PSBET-b vector contains argU gene, which encodes a rare Escherichia coli tRNA(Arg)(AGA/AGG). The argU gene helps A. aeolicus LeuRS, which contains AGA/AGG codons in exceptionally high frequency, express well in E. coli. The gene encoding beta subunit was inserted into pET-15b vector. E. coli BL21-CodonPlus (DE3) cells were transformed with the two recombinant plasmids to produce alphabeta-LeuRS with a His6 tag at the N-terminus of alpha subunit. The enzyme was purified by affinity chromatography on Ni-NTA Superflow. About 7 mg purified alphabeta-LeuRS was obtained from 250 ml culture. The His6-tag at the N-terminus did not affect the aminoacylation activity of the enzyme.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase beta-subunit C-terminal mutants.

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimeric enzyme that catalyzes post-translational farnesylation of many cellular regulatory proteins including p21 Ras. To facilitate the construction of site-directed mutants, a novel translationally coupled, two-cistron Escherichia coli expression system for rat FPT has been developed. This expression system enabled yields of >5 mg of purified protein per liter of E.coli culture to be obtained. The E.coli-derived FPT demonstrated an activity comparable to that of protein isolated from other sources. The reported expression system was used to construct three beta-subunit C-terminal truncation mutants, Delta5, Delta10 and Delta14, which were designed to eliminate a lattice interaction between the beta-subunit C-terminus of one molecule and the active site of a symmetry-related molecule. Steady-state kinetic analyses of these mutants showed that deletion up to 14 residues at the C-terminus did not reduce the value of kcat; however, Km values for both peptide and FPP increased 2-3-fold. A new crystalline form of FPT was obtained for the Delta10 C-terminal mutant grown in the presence of the substrate analogs acetyl-Cys-Val-Ile-Met-COOH peptide and alpha-hydroxyfarnesylphosphonic acid. The crystals diffract to beyond 2.0 A resolution. The refined structure clearly shows that both substrate analogs adopt extended conformations within the FPT active site cavity.     

Hepatitis C virus entry and the tetraspanin CD81

CD81, a member of the tetraspanin integral membrane protein family, has been identified as an essential receptor for HCV (hepatitis C virus). The present review highlights recent published data on the role that CD81 plays in HCV entry, including the importance of actin-dependent lateral diffusion of CD81 within the cell membrane, CD81 endocytosis and the CD81-Claudin-1 receptor complex in HCV internalization. Additional functions for CD81 in the viral life cycle and the role of HCV-CD81 interactions in HCV-induced B-cell and CNS (central nervous system) abnormalities are discussed.     

Full mass spectrometric characterization of human monoacylglycerol lipase generated by large-scale expression and single-step purification

The serine hydrolase monoacylglycerol lipase (MGL) modulates endocannabinoid signaling in vivo by inactivating 2-arachidonoylglycerol (2-AG), the main endogenous agonist for central CB1 and peripheral CB2 cannabinoid receptors. To characterize this key endocannabinoid enzyme by mass spectrometry-based proteomics, we first overexpressed recombinant hexa-histidine-tagged human MGL (hMGL) in Escherichia coli and purified it in a single chromatographic step with high yield (approximately 30 mg/L). With 2-AG as substrate, hMGL displayed an apparent V max of 25 micromol/(microg min) and K m of 19.7 microM, an affinity for 2-AG similar to that of native rat-brain MGL (rMGL) (Km=33.6 microM). hMGL also demonstrated a comparable affinity (Km approximately 8-9 microM) for the novel fluorogenic substrate, arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE), in a sensitive, high-throughput fluorometric MGL assay. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) unequivocably demonstrated the mass (34,126 Da) and purity of this hMGL preparation. After in-solution tryptic digestion, hMGL full proteomic characterization was carried out, which showed (1) an absence of intramolecular disulfide bridges in the functional, recombinant enzyme and (2) the post-translational removal of the enzyme's N-terminal methionine. Availability of sufficient quantities of pure, well-characterized hMGL will enable further molecular and structural profiling of this key endocannabinoid-system enzyme.     

High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris

Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.     

Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b

Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22(phox) as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.     

Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.     

Cloning, expression, purification, and characterization of the membrane protein UncI from Escherichia coli

The Escherichia coli unc-operon encodes the genes for the subunits of the F0F1-ATP synthase and an integral membrane protein of unknown function called UncI. UncI influences the cell-growth and activity of F0F1, but its exact function is still unknown. The expression level is too low to extract milligram amounts of UncI from E. coli membranes and the existing purification protocol based on methanol/chloroform is not suitable for structural and functional studies. Here we present protocols to increase the expression level, to purify UncI in a detergent where UncI is monodisperse, and we characterize its oligomeric state.     

Down-regulation of CD81 tetraspanin in human cells producing retroviral-based particles: tailoring vector composition

Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by reducing the amounts of the pseudotyped envelope and/or by incorporating protein capable of inducing immune responses when non-human VPC are used. Manipulating the repertoire of VPC proteins integrated onto the vector structure is an underexplored territory and should provide valuable insights on potential targets to improve vector pharmacokinetic and pharmacodynamic properties. In this work, human HEK 293 cells producing retrovirus-like particles (rVLPs) and infectious RV vectors were used to prove the concept of customizing RV composition by manipulating cellular protein content. The tetraspanin CD81 was chosen since it is significantly incorporated in the RV membrane, conferring to the vector significant immunogenicity when used in mice. RNA interference-mediated by shRNA lentiviral vector transduction was efficiently used to silence CD81 expression (up to 99%) and the rVLPs produced by knocked-down cells lack CD81. Silenced clones were analyzed for cell proliferation, morphological changes, susceptibility to oxidative stress conditions, and rVLP productivities. The results showed that the down-regulation of VPC proteins requires close monitoring for possible side effects on cellular production performance. Yet, they confirm that it is possible to change the composition of host-derived immunogens in RV by altering cellular protein content with no detriment for vector productivity and titers. This constitutes an important manipulation tool in vaccinology--by exploiting the potential adjuvant effect of VPC proteins or using them as fusion agents to other proteins of interest to be exposed on the vector membrane--and in gene therapy, by reducing the immunogenicity of RV-based vector and enhancing in vivo half-life. Such tools can also be applied to lentiviral or other enveloped viral vectors.     

High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase.

The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.     

High-level expression of recombinant active human renin in Sf-9 cells: rapid purification and characterization

Recombinant human (rh) renin was expressed in Sf-9 insect cells. Baculovirus-infected Sf-9 cells produced active rh-renin in the late stage of cultivation. The rh-renin was purified after 5 d of culture by two steps of column chromatography. Approximately 0.61 mg of pure rh-renin was obtained from 200 ml of culture medium with a yield of 35.3%.     

The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.     

High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.