Protein and Peptide Letters - Volume 14, Issue 5, 2007

The cysteine-rich N-terminal domain of the micronemal adhesive protein MIC1 (MIC1-NT) from Toxoplasma gondii was cloned, expressed in Escherichia coli and purified. MIC1-NT is amenable to structural studies as shown by preliminary NMR and X-ray analysis. Positive results with two further micronemal proteins indicate that our strategy has wider application.

Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirusinfected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37°C, but increased to 85% at 22°C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBPfused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.

Protein HC is a low molecular weight heterogeneous glycoprotein widely distributed in human body fluids and belonging to the lipocalin superfamily. The monomer contains a single (183 amino acid residues long) peptide chain with 3 cysteine residues (2 of which form a disulfide bridge) and is glycosylated. The molecular mass of the glycosylated protein is about 27 kDa. Native gel electrophoresis results revealed partial oligomerisation of protein HC, which therefore was analysed by gel filtration. Two forms (monomer and dimer) of the protein HC were isolated. The SAXS data were recorded on an X33 camera using synchrotron radiation (λ=0.15 nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg, Germany). Solution scattering results permitted determination of the structural parameters of both forms of the protein studied. The monomer of protein HC is characterised by a radius of gyration RG= 2.20 nm and Dmax=6.3 nm and the dimer by RG=2.99 nm and Dmax=9.5 nm.

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.

A novel disintegrin, stejnin, was purified from the Trimeresurus stejnegeri venom by gel filtration and reverse phase high performance liquid chromatography. The molecular weight of stejnin was determined to be 7428 Da by MALD-TOF MS analysis. The cDNA encoding the precursor of stejnin was cloned from the venom gland. From the deduced amino acid sequence, stejnin is composed of 71 amino acid residues contains the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Stejnin strongly inhibited ADP- and ristomycin-induced human platelet aggregation with IC50 of 45 and 50 nM, respectively. Stejnin also possessed potent inhibited cell proliferation of ECV304 cells.

The Surface Plasmon Resonance Imaging (SPRI) sensor for papain determination based on the interaction between the immobilized cystatin and aqueous papain solution has been developed. The development of the sensor is a stage in the development of the sensor for the determination of cystein proteinases of the papain group. Cystatin was immobilized onto a gold chip by means of a thiol underlayer (cysteamine) and EDS/NHS reaction. Conditions of cystatin- papain interaction were optimized (pH, time of interaction and cystatin concentration). The developed sensor works within the range of 1 - 10 ng ml-1. However, the precision of measurements (RSD equal to 45%) should be improved. The response of the sensor to papain is specific. This was checked using BSA as a reference.

In this study, peptides and proteins extracted from string bean with 1% TCA were separated by chromatography on columns with copper ions immobilized through IDA and o-phosphoserine OPS. Protein and peptide concentrations and the anti-free-radical properties of the isolated fractions were determined. Identification was obtained using mass spectrometry. The anti-free- radical activity of all analyzed samples was determined using the DPPH· test, and was found to depend on reaction time, choice of chelating agent and the order in which the fractions were eluted from the column.

The immunogenicity of “novel” MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase141-161 peptide was found to be immunogenic and endogenously processed in the HLA-DRβ1*0101 and HLADR β1*0401 transgenic mice with peptide specific production of IFNγ or IL-5 respectively. The MART-129-43 peptide was only found immunogenic in HLA-DRβ1*0101 mice.

Benzimidazole resistance in thirty isolates of the biocontrol agents was studied with reference to specific mutations in the β-tubulin genes. Our results suggest that apart from the correlation between specific mutations in the β-tubulin gene and variation in resistance, the overall ratio of polar and non-polar amino-acids may also play a vital role in conferring benzamidazole resistance to these fungi.

In this study, we attempt to improve the prediction of mutation position in H5N1 hemagglutinin from influenza A virus using the neural network model with distinguishing of arginine, leucine and serine. In comparison with previous logistic regression prediction model, the 3-6-1 feedforward backpropagation neural network model reduced independents from seven to three without compromising the predictability, and the prediction is made according to the most similar parameterised hemagglutinin rather than the population means.

Symmetrical peptide GYDTQAIVENNESTEYG (WT, Wild Type) identical to 35-51 aminoacid residues of human alpha-lactalbumin (HLA) and peptide GYDTQTVVNNNGHTDYG (ID, IDeal symmetry) homologous to betadomain of mammalian alpha-lactalbumins can form amyloid-like fibrils in conditions required for fibrillogenesis of HLA. The latter peptide can also form fibrils in deionized water. Fibrils formed by these peptides can cause forming of HLA amyloid-like aggregates in physiological conditions. These results provide an evidence for presence of amyloidogenic determinant in beta-domain of alpha-lactalbumin. Thus, symmetry in the primary structure may play the role in fibrillogenesis of proteins.

The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.

Blue oxygen binding protein hemocyanin from scorpion Buthus sindicus has been investigated using low resolution techniques. The native protein is a polymer of eight different types of subunits arranged in four cubic hexameric form (4x6-mers) as previously annotated using a combination of various types of chromatographic and electrophoretic techniques. In addition, both “top face” as well as the “side view” of the native assembly has also been identified from the negatively stained specimens using transmission electron microscopy confirming the overall structural features of arthropodan hemocyanins. These results are also supported from data obtained from another low resolution technique i.e. Small Angle X-ray scattering (SAXS). SAXS results under oxygenated and deoxygenated states represent a validation case for this technique with key conformational changes of Rg 88.0 → 86.0 Å ± 1% (Dmax 280.0 → 290.0 Å ± 2%), respectively suggesting that the oxygenated hemocyanin is longer then the deoxygenated hemocyanin by almost 2 Å. Likewise, active conformations of the purified structural and functional subunit Bsin1 under oxygenated and deoxygenated states also determined by SAXS measurements revealed a Rg value of 25.2 → 25.7 Å ± 1% (Dmax 75.0 → 75.5 Å ± 2%), respectively suggesting very little or no contribution of the individual subunit in the overall conformational change in the native assembly during molecular breathing. Preliminary molecular shapes for the oxy-molecules, calculated directly from the scattering profile-alone in a model-independent procedure, superimpose well on other closely related known threedimensional structures of the same size. Structural and functional aspects of the native as well as purified subunit and the application of these low resolution techniques like transmission electron microscopy and Small Angle X-ray scattering have been discussed.

The detection and identification of O-phosphorylation sites in proteins with mass spectrometry remains a challenge. A common approach to analyse these modifications is to enrich phosphopeptides by immobilized metal affinity chromatography (IMAC) prior to mass spectrometric analysis. In this study two commercially available IMAC kits based on Fe(III)-ions immobilized on magnetic beads and Ga(III)-ions immobilized on a chelate-resin, have been investigated and the binding efficiency of peptide mixtures containing non-phosphorylated, singly, doubly and triply phosphorylated peptides have been tested.

Peptide analogs of tendamistat were synthesized and analyzed for α-amylase inhibitory activity. The pKa of the N-terminal tyrosine was modified by incorporation of ring-substituted analogs, which alters hydrogen bonding capacity. Ki values ranging from 70 to 524 μM generally increased with increasing pKa, indicating a necessity for H-bond donor ability.

The smu.134 gene encodes a putative transcriptional regulator of 217 residues in Streptococcus mutans, a major pathogen for human dental caries. The gene was cloned into expression vector pET28?? and expressed in soluble form in E. coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.134 was purified to homogeneity in a two step procedure of Ni2+ chelating and size exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by hanging-drop vapor diffusion method and diffracted to 2.6 Å. The crystal belonged to space group P212121, with unit-cell parameters a=55.03 Å, b=80.84 Å, c=107.96 Å.

Sau3AI is a type II restriction endonuclease that recognizes the palindromic sequence 5’-GATC-3’ and cleaves 5’ to G residue on each strand. The E64A mutant full length protein was cloned and expressed in Escherichia coli. The purified (His)6-tagged protein has monomer and dimer fraction and was crystallized by the hanging-drop vapor-diffusion technique. The dimer protein crystals can diffract to 3.0Å resolution and the monomer protein crystals can diffract to better than 2.8Å resolution. One completed dataset has been collected and it shows that the monomer orthorhombic Sau3AI/E64A crystal is in space group C2221 with unit cell parameters (69.44, 197.60, 191.46, 90, 90, 90) and contains two molecules in one asymmetric unit.