Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Šbetween the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

Structural and functional characterization of a novel lipolytic enzyme from a Brazilian Cerrado soil metagenomic library.

OBJECTIVE: To isolate putative lipase enzymes by screening a Cerrado soil metagenomic library with novel features. RESULTS: Of 6720 clones evaluated, Clone W (10,000 bp) presented lipolytic activity and four predicted coding sequences, one of them LipW. Characterization of a predicted esterase/lipase, LipW, showed 28% sequence identity with an arylesterase from Pseudomonas fluorescens (pdb|3HEA) from protein database (PDB). Phylogenetic analysis showed LipW clustered with family V lipases; however, LipW was clustered in different subclade belonged to family V, suggesting a different subgroup of family V. In addition, LipW presented a difference in family V GH motif, a glycine replaced by a serine in GH motif. Estimated molecular weight and stokes radius values of LipW were 29,338.67-29,411.98 Da and 2.58-2.83 nm, respectively. Optimal enzyme activity was observed at pH 9.0-9.5 and at 40 °C. Circular dichroism analysis estimated secondary structures percentages as approximately 45% α-helix and 15% ß-sheet, consistent with the 3D structure predicted by homology. CONCLUSION: Our results demonstrate the isolation of novel family V lipolytic enzyme with biotechnological applications from a metagenomic library.

Crystal structure of VapC21 from Mycobacterium tuberculosis at 1.31 Å resolution.

Persisters are individual bacterial cells that exhibit a phenotype characterized by slow growth, low metabolic rate and multidrug tolerance. The processes that drive cells into a persistence state constitute an active but incipient research field, and structural data regarding its components are scarce. The molecular targets of many therapeutic drugs are involved in cell wall synthesis and cell division, and these cellular processes are down-regulated in persister cells, consequently these cells are more likely to survive antibiotic treatment. Toxin-antitoxin systems were shown to have a leading role in the formation of persisters, and several pathogenic bacteria display a wide array of these systems. The Mycobacterium tuberculosis H37Rv genome presents 88 toxin-antitoxin loci, of which 47 code for members of the VapBC protein family. To date, only four crystal structures of Mycobacterium tuberculosis VapBC complexes are available, and all of them present the toxin bound to and inhibited by the antitoxin. We present the 1.31 Å resolution structure of VapC21, the first structure of a Mycobacterium tuberculosis VapC toxin in the absence of its cognate inhibitory antitoxin. Our data show that VapC21 is a dimer in solution, with conserved active site architecture and an extensive antitoxin binding groove. Additionally, the strategy used to mutate a putative catalytic residue allowing the expression and purification of soluble VapC21 will pave the way for the resolution of more toxin structures in the absence of antitoxins. Taken together, our findings represent an important step in unraveling the molecular mechanisms related to persistence, which will contribute for the design of faster and more efficient therapeutic approaches for the treatment of tuberculosis, particularly for infections with multidrug-resistant strains.

Structural and functional characterization of the recombinant thioredoxin reductase from Candida albicans as a potential target for vaccine and drug design.

The thioredoxin system plays a critical role in maintaining the cytoplasm redox state, participating in functions that are important to the cellular viability of fungi. Although functional and structural information on targets in human pathogenic fungi has been scarcely described in the literature, such studies are essential for in silico drug design and biotechnological applications. Therefore, the aims of the present study were to produce recombinant proteins of the thioredoxin system from Candida albicans and evaluate their possible use as prophylactic or alternative therapies against the most important pathogenic fungus associated with nosocomial infections. We focused on biochemical and structural analyses of recombinant thioredoxin reductase from C. albicans with His-tag (CaTrxR-His) for further biotechnology applications. Heterologous CaTrxR-His was efficiently expressed in the soluble fraction of the Escherichia coli lysate. CaTrxR-His was obtained with a high level of purity and presented specific enzymatic activity. Conformational changes of the protein were observed at different pHs and temperatures, with higher thermal stability at pH 8.0. The CaTrxR-His vaccine was shown to effectively induce high levels of CaTrxR-specific immunoglobulin G antibodies in Balb/c mice and reduce the renal fungal burden of experimental disseminated candidiasis in mice. These data may greatly impact future development strategies for vaccine and drug designs against C. albicans infection.

Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

Crystal structure and interconversion of monomers and domain-swapped dimers of the walnut tree phytocystatin.

Biotechnological applications of phytocystatins have garnered significant interest due to their potential applications in crop protection and improve crop resistance to abiotic stress factors. Cof1 and Wal1 are phytocystatins derived from Coffea arabica and Juglans regia, respectively. These plants hold significant economic value due to coffee's global demand and the walnut tree's production of valuable timber and widely consumed walnuts with culinary and nutritional benefits. The study involved the heterologous expression in E. coli Lemo 21(DE3), purification by immobilized metal ion affinity and size exclusion chromatography, and biophysical characterization of both phytocystatins, focusing on isolating and interconverting their monomers and dimers. The crystal structure of the domain-swapped dimer of Wal1 was determined revealing two domain-swapped dimers in the asymmetric unit, an arrangement reminiscent of the human cystatin C structure. Alphafold models of monomers and Alphafold-Multimer models of domain-swapped dimers of Cof1 and Wal1 were analyzed in the context of the crystal structure. The methodology and data presented here contribute to a deeper understanding of the oligomerization mechanisms of phytocystatins and their potential biotechnological applications in agriculture.

An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans.

BACKGROUND: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking. RESULTS: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and ß-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports. CONCLUSIONS: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.

Ts17, a Tityus serrulatus ß-toxin structurally related to α-scorpion toxins.

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Nav1.1-Nav1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na+-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Nav1.2 and Nav1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Nav1.5. Circular dichroism spectra showed that Ts17 presents mainly ß-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug. Key contribution Purification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, ß-toxin classification.

Structure and function of cationic hylin bioactive peptides from the tree frog Boana pulchella in interaction with lipid membranes.

Amphibians have a great diversity of bioactive peptides in their skin. The cDNA prepro-peptide sequencing allowed the identification of five novel mature peptides expressed in the skin of Boana pulchella, four with similar sequences to hylin peptides having a cationic amphipathic-helical structure. Whole mature peptides and some of their fragments were chemically-synthesized and tested against Gram-positive and Gram-negative bacterial strains. The mature peptide hylin-Pul3 was the most active, with a MIC= 14 µM against Staphylococcus aureus. Circular dichroism assays indicated that peptides are mostly unstructured in buffer solutions. Still, adding large unilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoylphosphatidylglycerol increased the α-helix content of novel hylins. These results demonstrate the strong influence of the environment on peptide conformation and highlight its significance while addressing the pharmacology of peptides and their biological function in frogs.

Purification and Biological Properties of Raniseptins-3 and -6, Two Antimicrobial Peptides from Boana raniceps (Cope, 1862) Skin Secretion.

The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans' skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.

rMELEISH: A Novel Recombinant Multiepitope-Based Protein Applied to the Serodiagnosis of Both Canine and Human Visceral Leishmaniasis.

BACKGROUND: visceral leishmaniasis (VL) is a critical public health problem in over ninety countries. The control measures adopted in Brazil have been insufficient when it comes to preventing the spread of this overlooked disease. In this context, a precise diagnosis of VL in dogs and humans could help to reduce the number of cases of this disease. Distinct studies for the diagnosis of VL have used single recombinant proteins in serological assays; however, the results have been variable, mainly in relation to the sensitivity of the antigens. In this context, the development of multiepitope-based proteins could be relevant to solving such problem. METHODS: a chimeric protein (rMELEISH) was constructed based on amino acid sequences from kinesin 39 (k39), alpha-tubulin, and heat-shock proteins HSP70 and HSP 83.1, and tested in enzyme-linked immunosorbent (ELISA) for the detection of L. infantum infection using canine (n = 140) and human (n = 145) sera samples. RESULTS: in the trials, rMELEISH was able to discriminate between VL cases and cross-reactive diseases and healthy samples, with sensitivity and specificity values of 100%, as compared to the use of a soluble Leishmania antigenic extract (SLA). CONCLUSIONS: the preliminary data suggest that rMELEISH has the potential to be tested in future studies against a larger serological panel and in field conditions for the diagnosis of canine and human VL.

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicola grisea xylanase.

Humicola grisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichia pastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40-50 °C and pH 5.0-9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

Crystal structure and physicochemical characterization of a phytocystatin from Humulus lupulus: Insights into its domain-swapped dimer.

Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.

Figainin 1, a Novel Amphibian Skin Peptide with Antimicrobial and Antiproliferative Properties.

Amphibian skin secretions are abundant in bioactive compounds, especially antimicrobial peptides. These molecules are generally cationic and rich in hydrophobic amino acids, have an amphipathic structure and adopt an α-helical conformation when in contact with microorganisms membranes. In this work, we purified and characterized Figainin 1, a novel antimicrobial and antiproliferative peptide from the cutaneous secretion of the frog Boana raniceps. Figainin 1 is a cationic peptide with eighteen amino acid residues-rich in leucine and isoleucine, with an amidated C-terminus-and adopts an α-helical conformation in the presence of trifluoroethanol (TFE). It displayed activity against Gram-negative and especially Gram-positive bacteria, with MIC values ranging from 2 to 16 µM, and showed an IC50 value of 15.9 µM against epimastigote forms of T. cruzi; however, Figanin 1 did not show activity against Candida species. This peptide also showed cytolytic effects against human erythrocytes with an HC50 of 10 µM, in addition to antiproliferative activity against cancer cells and murine fibroblasts, with IC50 values ranging from 10.5 to 13.7 µM. Despite its adverse effects on noncancerous cells, Figainin 1 exhibits interesting properties for the development of new anticancer agents and anti-infective drugs against pathogenic microorganisms.

Blood pressure-lowering effects of a Bowman-Birk inhibitor and its derived peptides in normotensive and hypertensive rats.

Bioactive plant peptides have received considerable interest as potential antihypertensive agents with potentially fewer side effects than antihypertensive drugs. Here, the blood pressure-lowering effects of the Bowman-Birk protease inhibitor, BTCI, and its derived peptides, PepChy and PepTry, were investigated using normotensive (Wistar-WR) and spontaneously hypertensive rats (SHR). BTCI inhibited the proteases trypsin and chymotrypsin, respectively, at 6 µM and 40 µM, a 10-fold greater inhibition than observed with PepTry (60 µM) and PepChy (400 µM). These molecules also inhibited angiotensin converting enzyme (ACE) with IC50 values of 54.6 ± 2.9; 24.7 ± 1.1; and 24.4 ± 1.1 µM, respectively, occluding its catalytic site, as indicated by molecular docking simulation, mainly for PepChy and PepTry. Gavage administration of BTCI and the peptides promoted a decrease of systolic and diastolic blood pressure and an increase of renal and aortic vascular conductance. These effects were more expressive in SHR than in WR. Additionally, BTCI, PepChy and PepTry promoted coronary vasodilation and negative inotropic effects in isolated perfused hearts. The nitric oxide synthase inhibitor blunted the BTCI and PepChy, with no cardiac effects on PepTry. The findings of this study indicate a therapeutic potential of BTCI and its related peptides in the treatment of hypertension.

Biological Properties of a Novel Multifunctional Host Defense Peptide from the Skin Secretion of the Chaco Tree Frog, Boana raniceps.

In recent years, the number of new antimicrobial drugs launched on the market has decreased considerably even though there has been an increase in the number of resistant microbial strains. Thus, antimicrobial resistance has become a serious public health problem. Amphibian skin secretions are a rich source of host defense peptides, which generally are cationic and hydrophobic molecules, with a broad-spectrum of activity. In this study, one novel multifunctional defense peptide was isolated from the skin secretion of the Chaco tree frog, Boana raniceps. Figainin 2 (1FLGAILKIGHALAKTVLPMVTNAFKPKQ28) is cationic and hydrophobic, adopts an α-helical structure in 50% (v/v) trifluoroethanol (TFE), and is thermally stable. This peptide exhibited activity against Gram-negative and Gram-positive pathogenic bacteria arboviruses, T. cruzi epimastigotes; however, it did not show activity against yeasts. Figainin 2 also showed antiproliferative activity on cancer cells, is moderately active on human erythrocytes, and activates the oxidative burst in human neutrophils.

Xylanase from Aspergillus tamarii shows different kinetic parameters and substrate specificity in the presence of ferulic acid.

A 22 kDa xylanase (AtXyl1) from Aspergillus tamarii was purified by two chromatographic steps and presented preference for oat spelt (OSX), birchwood (BrX) and beechwood (BeX) xylans respectively, as substrates. AtXyl1 displays the highest activity at pH 5.5 and 55 °C and showed tolerance over a range of different phenolic compounds. The activity of AtXyl1 was not inhibited when the enzyme was incubated with ferulic acid (FA) using OSX or BrX as substrate. On the other hand, the incubation of AtXyl1 with BeX and FA resulted in an increase in enzyme activity. The molecular docking of a GH11 xylanase from Aspergillus niger with FA showed the preference for binding within the catalytic site. The position of FA was based on the presence or absence of a complexed substrate. When the enzyme from A. niger was docked in the absence of xylan in its crystal structure, FA interacted with Tyr164 and a water molecule. For the enzyme socked with xylo-oligosaccharides, FA interacted with Ser94, Tyr89 and the xylo-oligosaccharide present in the catalytic site. Thermodynamic parameters from the reaction of AtXyl1 with different xylans and FA indicate that FA can cause a conformational change in the enzyme, and this can influence the substrate fitting and makes the enzyme tolerant or active toward the substrate. Our findings suggest that enzyme activation or tolerance to phenolic compounds can be correlated to subtle changes in enzyme conformation due to the presence of the phenolic compound.

Biochemical and structural characterization of a protein complex containing a hyaluronidase and a CRISP-like protein isolated from the venom of the spider Acanthoscurria natalensis.

Spider venoms are composed of a complex mixture of bioactive molecules. The structural and functional characterization of these molecules in the venom of the Brazilian spider Acanthoscurria natalensis, has been little explored. The venom was fractionated using reversed-phase liquid chromatography. The fraction with hyaluronidase activity was named AnHyal. The partial sequencing of AnHyal revealed the presence of a CRISP-like protein, in addition to hyaluronidase, comprising 67% coverage for hyaluronidase from Brachypelma vagans and 82% for CRISP-like protein from Grammostola rosea. 1D BN-PAGE zymogram assays of AnHyal confirmed the presence of enzymatically active 53 kDa monomer and 124 and 178 kDa oligomers. The decomposition of the complexes by 2D BN/SDS-PAGE zymogram assays showed two subunits, 53 (AnHyalH) and 44 kDa (AnHyalC), with sequence similarity to hyaluronidase and CRISP proteins, respectively. The secondary structure of AnHyal is composed by 36% of α-helix. AnHyal presented maximum activity at pH between 4.0 and 6.0 and 30 and 60 °C, showed specificity to hyaluronic acid substrate and presented a KM of 617.9 µg/mL. Our results showed that hyaluronidase and CRISP proteins can form a complex and the CRISP protein may contribute to the enzymatic activity of AnHyalH.

Purification, crystallization and preliminary crystallographic studies of SPCI-chymotrypsin complex at 2.8 A resolution.

A binary complex of the Schizolobium parahyba chymotrypsin inhibitor (SPCI) with chymotrypsin was purified by size-exclusion chromatography and crystallized by the sitting-drop vapour-diffusion method with 100 mM MES-NaOH pH 5.5, 20%(w/v) PEG 6000, 200 mM LiCl as precipitant and 200 mM nondetergent sulfobetaine molecular weight 201 Da (NDSB-201) as an additive. SPCI is a small protein with 180 amino-acid residues isolated from S. parahyba seeds and is able to inhibit chymotrypsin at a 1:1 molar ratio by forming a stable complex. X-ray data were collected to 2.8 A resolution from a single crystal of the SPCI-chymotrypsin binary complex under cryogenic conditions. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.28, b = 64.57, c = 169.23 A, and the R(merge) is 0.122 for 11 254 unique reflections. A molecular-replacement solution was found using the preliminary crystal structure of SPCI and the structure of chymotrypsin (PDB code 4cha) independently as search models.