Recombinant protein purification in baculovirus-infected Rachiplusia nu larvae: An approach towards a rational design of downstream processing strategies based on chromatographic behavior of proteins.

Expression of recombinant proteins with baculovirus-infected insect larvae is a scarcely investigated alternative in comparison to that in insect cell lines, a system with growing popularity in the field of biotechnology. The aim of this study was to investigate the chromatographic behavior and physicochemical properties of the proteome of Rachiplusia nu larvae infected with recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), in order to design rational purification strategies for the expression of heterologous proteins in this very complex and little-known system, based on the differential absorption between target recombinant proteins and the system's contaminating ones. Two-dimensional (2D) gel electrophoresis showed differences in the protein patterns of infected and non-infected larvae. Hydrophobic interaction matrices adsorbed the bulk of larval proteins, thus suggesting that such matrices are inappropriate for this system. Only 0.03% and 2.9% of the total soluble protein from the infected larval extract was adsorbed to CM-Sepharose and SP-Sepharose matrices, respectively. Immobilized metal ion affinity chromatography represented a solid alternative because it bound only 1.4% of the total protein, but would increase the cost of the purification process. We concluded that cation-exchange chromatography is the best choice for easy purification of high-isoelectric-point proteins and proteins with arginine tags, since very few contaminating proteins co-eluted with our target protein.

Structural glance into a novel anti-staphylococcal peptide.

Novel antimicrobial peptides are valuable molecules for developing anti-infective drugs to counteract the contemporary spread of microbial drug-resistance. Here we focus on a novel peptide (RKWVWWRNR-NH2) derived from the fragment 107-115 of the human lysozyme that displays a 20-fold increase in anti-staphylococcal activity. The conformational analysis of this peptide and its interaction with model lipidic phases-as assayed by circular dichroism and fluorescence spectroscopy-show a noteworthy spectral change, which might be related to its anti-staphylococcal activity. The secondary structure of peptide [K(108)W(111)] 107-115 hLz was dramatically affected through a single substitution at position 111 (Ala by Trp). Therefore, this conformational change might improve the interaction of the novel peptide with the bacterial plasma membrane. These results highlight the role of peptide secondary structure and the distribution of polar/nonpolar residues for the effective interaction of this peptide with the bacterial plasma membrane, a key step for triggering its lethal effect. This knowledge may contribute to the rational design of a new generation of antimicrobial peptides with increased efficacy developed from natural sources by simple screening tools.

Design of Affinity Chromatography Peptide Ligands Through Combinatorial Peptide Library Screening.

In this chapter, a protocol to design affinity chromatography matrices with short peptide ligands immobilized for protein purification is described. The first step consists of the synthesis of a combinatorial peptide library on the hydroxymethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry. Next, the library is screened with the protein of interest labeled with a fluorescent dye or biotin. Subsequently, peptides contained on positive beads are identified by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), and those sequences showing greater consensus are synthesized in larger quantities and immobilized on chromatographic supports. Finally, target protein adsorption on peptide affinity matrices is evaluated through equilibrium adsorption isotherms and breakthrough curves.

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep).

AIMS: Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease-catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent-tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. METHODS AND RESULTS: The tripeptide Ac-Phe-Gly-Phe-NH(2) was synthesized using Ac-Phe-OEt and Gly-Phe-NH(2) substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1.5 or 0.5 mol l(-1) NaCl. Purification and identification of the peptide product was achieved by RP-HPLC and ESI-MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. CONCLUSION: Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. SIGNIFICANCE AND IMPACT OF STUDY: The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.

Two-step enzymatic strategy for the synthesis of a smart phenolic polymer and further immobilization of a ß-galactosidase able to catalyze transglycosydation reaction.

A rapid and efficient enzymatic procedure for the preparation of an immobilized ß-galactosidase has been described. In a first step, soybean peroxidase was used to catalyze the polymerization of a strategically activated phenol (N-Succinimidyl 3-(4-hydroxyphenyl)propionate, known as Bolton-Hunter reagent). The phenolic support was directly employed for immobilizing ß-galactosidase from Bacillus circulans (ATCC 31382, ß-Gal-3), giving rise to a new biocatalyst subsequently applied in the synthesis of a ß-galatodisaccharide (Gal-ß(1-3)-GlcNAc and Gal-ß(1-3)-GalNAc). The reaction proceeded with high conversion rates and total regioselectivity. Reusability assays were performed with the same reaction conditions finding that the immobilized enzyme retains about 55% of its activity after eight batches. Finally and based on our results, the two-step enzymatic procedure presented here is a good and green alternative to the preparation of carbohydrates with biological activities.

Purification and characterization of two forms of antigen 5 from polybia scutellaris venom.

Two polypeptides from the venom of Polybia scutellaris were purified to homogeneity by RP-HPLC. They differ very slightly in mol. wt (both are about 23,000) and hydrophobicity, and have isoelectric points greater than 9. Amino acid analyzes show close similarity between them and with antigen 5 of vespids from different species. The two polypeptides have an identical N-terminal sequence (18 amino acids) which shows a high degree of homology with those of other vespids. Owing to the fact that the venom of this species is non-allergenic, the data for the mol. wt, isoelectric point, amino acid composition and N-terminal sequence allow us to identify the isolated polypeptides as two forms of antigen 5. Amino acids at positions 5 and 11 in P. scutellaris antigen 5 differ from those of the previously known sequences for antigen 5, suggesting that one or other might be responsible for the lack of allergenicity of the P. scutellaris venom.

Foot and mouth disease virus concentration and purification by affinity chromatography.

Foot and mouth disease virus, (FMDV) from a crude cell lysate was purified in a single step by affinity chromatography with heparin as a ligand. The virus eluted from an Heparin-Ultrogel A4R column at 1M sodium chloride in 10 mM sodium phosphate buffer, pH 7.0, while most cell protein and albumin did so at lower concentrations of sodium chloride in the same buffer. Purity of the eluted fraction containing the virus was assessed by SDS-PAGE, HPLC, ultracentrifugation, and UV absorption spectrum. With this method, intact viral particles are recovered in high yield (over 90%) and specific virus purity increases nearly 1000-fold. The capacity of the chromatographic matrix for the virus was found to be 1.1 mg viral mass per mL of hydrated gel.

Purification and characterization of a milk clotting protease from Mucor bacilliformis.

An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.

Solid state production of a Mucor bacilliformis acid protease.

Acid protease production by a local strain of Mucor bacilliformis was performed by solid state cultivation on different agricultural by-products as substrate. The effects of different parameters on enzyme biosynthesis were studied: Wheat bran wetted at 120% with a 200 mM HCl solution and inoculated with 5 x 10(5) spores/g produced a milk clotting activity of 7500 U/g bran after 72 h cultivation at 24 degrees C.

Optimization by factorial analysis of caprylic acid precipitation of non-immunoglobulins from hyperimmune equine plasma for antivenom preparation.

Optimization of caprylic acid precipitation of equine plasma non-immunoglobulin proteins for antivenom preparation was achieved by regression analysis of the responses of three highly significant factors assayed by factorial design. The factors studied were caprylic acid concentration, plasma pH and temperature, and their response was assessed in terms of filtration speed, residual albumin, total protein content and turbidity. The results evidenced that the three variables are involved in the precipitation process. Moreover, the factors displayed significant interactions, indicating that their levels distinctly affect the optimization procedure. The best combination was 3% caprylic acid, 37 °C and plasma pH 4.9; under these conditions, all immunoglobulins and only 0.1% albumin remained in the supernatant, in a very fast and simple procedure. After formulation, the antivenom obtained by this procedure presented full lethality neutralizing activity and absence of protein aggregates.

Recombinant peroxidase production in species of lepidoptera frequently found in Argentina.

Horseradish peroxidase isozyme C (HRPC) is an important commercial biocatalyst. In this study, a screening of different lepidopteran species frequently found in Argentina to produce this protein was carried out. Two recombinant viruses were constructed: AcMNPV HRPC polyhedrin-minus (occ-), an intrahemocelical infective virus; and AcMNPV HRPC polyhedrin-plus (occ+), to achieve an oral infective baculovirus. Each lepidopteran species was infected either with AcMNPV HRPC occ- or AcMNPV HRPC occ+ and the harvesting days post-infection (dpi) were optimized. All species were susceptible to AcMNPV HRPC occ- infection, giving Spodoptera frugiperda the best yield: 41 µg per larva. Rachiplusia nu was highly susceptible to oral infection, reaching 22 µg per larva at 4 dpi. HRPC was purified by IMAC from S. frugiperda extracts with a yield of 86% and a purification factor of 29.

Rapid affinity purification processes for cyclodextrin glycosyltransferase from Bacillus circulans.

Two rapid and easy-to-scale-up methods for the purification of cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans were developed: affinity precipitation with starch and aqueous two-phase partition. The first method, optimised by a factorial design, gave an 80% CGTase adsorption at 11% starch and 1.6% ammonium sulphate, and a 65% recovery after elution with 10 mM alpha-cyclodextrin. The purification factor was 17. Aqueous two-phase partition yielded a 72% CGTase recovery in a two-step procedure; CGTase was obtained in the bottom phase with a purification factor of 37.

Content of thiophenes in transformed root cultures of argentinian species of tagetes.

The presence of thiophenes in four Argentinian species of TAGETES was studied. T. TERNIFLORA HBK and T. MINUTA L. seedlings contain 5-(4-hydroxy-1-butynyl)-2-2'-bithienyl (BBTOH); 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), while T. CAMPANULATA Griseb and T. LAXA Cabrera seedlings also accumulated BBT and alpha-T. From the four TAGETES species tested only T. LAXA was able to produce transformed roots when infected with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones of transformed roots were obtained in which the total thiophene content present showed considerable variations (277 to 1773 microg/g FW). The thiophene spectrum, however, was similar between different clones. In addition, the thiophene patterns in these transformed clones differed from that formed in the parent plants.

High-speed pectic enzyme fractionation by immobilised metal ion affinity membranes.

Immobilised metal ion affinity polysulfone hollow-fibre membranes, with a high capacity for protein adsorption, were prepared and their utilisation for commercial pectic enzyme fractionation was studied. The pass-through fraction containing pectinlyase is useful for fruit-juice clarification without methanol production on account of pectinesterase being retained by the IDA-Cu2+ membrane.

Effect of the surface charge on partitioning of chemically modified bovine serum albumin in aqueous two-phase systems.

In order to assess the influence of the protein charge on its partitioning in poly(ethyleneglycol)/salt aqueous two-phase systems, three bovine serum albumin derivatives with isoelectric points of 5.50, 6.20 and 6.85 were obtained by chemical modification of the protein with a soluble carbodiimide and glycine O-methyl ester and separation of the derivative mixture by liquid isoelectric focusing. The modification reaction was mild enough to preserve the tertiary structure of the proteins, as judged by circular dichroism and fourth derivative UV spectra. The surface hydrophobicity of the bovine serum albumin derivatives was identical, as measured by hydrophobic interaction chromatography. Partitioning of the derivatives in poly(ethyleneglycol)/phosphate and poly(ethyleneglycol)/citrate aqueous two-phase systems between pH 5.2 and 6.5 indicates that partitioning is not dependent on the protein charge in the poly(ethyleneglycol)/salt systems studied.

Ethoxyformylation of histidine residues in equine growth hormone.

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.

Molecular characterization of a protein, insoluble at low temperature, produced by Clostridium botulinum type G.

A preliminary study of a low-toxicity protein, called cryoprotein, produced by Clostridium botulinum type G, led to a better characterization of this substance and to discriminate its relationship with type G botulinum toxin. This sparingly soluble protein has been characterized as an aggregated form of a soluble precursor with an Mr of 170,000. This phenomenon is temperature-dependent. The monomeric protein is usually contaminated with a lower Mr form (150,000) quite probably originated by a limited proteolytic process. The amino acid composition of this protein is relatively analogous to that of the botulinum toxins A and B, the only notable exception being the absence of cysteine. The N-terminal amino acid is alanine and the C-terminal sequence is Val-Ala-Leu-OH. The low toxicity which is usually demonstrable in samples of this protein disappears after a reductive treatment, strongly suggesting that it is not an intrinsic property. Taking into account that some of its physiochemical properties are similar to those of the known botulinal toxins, it is quite probable that this substance accompanies G toxin preparations currently obtained by routine methods, increasing its non-toxic antigenic mass. This fact could be critical to the sensitivity and specificity of G toxin immunological detection methods.

Oxidation of methionine residues in equine growth hormone by Chloramine-T.

1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone. 2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved. 3. Methionine 4 is the most reactive group, followed by methionines 72 and 178--methionine 123 being the less reactive residue. 4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues. 5. Results agree with data previously obtained with bovine growth hormone.