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.

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.

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.

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.

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.

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.

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.

Fractionation of the naringinase complex from Aspergillus terreus by dye affinity chromatography.

Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).

Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries.

The peptide, Ala-Pro-Ala-Arg (APAR), was selected from the screening of a tetrapeptide combinatorial synthetic library as the ligand for affinity purification of an anti-Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) monoclonal antibody (Mab) developed in mouse ascitis. The affinity chromatographic matrix obtained by attachment of APAR to agarose, having a peptide density of 0.5 micromol ml(-1), showed a maximum capacity of 9.1 mg Mab ml(-1) and a dynamic capacity of 3.9 mg Mab ml(-1). A 95% yield of electrophoretically pure anti-GM-CSF was obtained in a single step.

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.

Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.

Protein adsorption onto tentacle cation-exchange hollow-fiber membranes

A sulfonic group (up to 200 &mgr;mol/mL) membrane was incorporated to epoxy-activated microporous hollow fibers to obtain high-capacity convective ion exchangers. The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly. At sulfonic group density of 70 &mgr;mol/mL, the membrane lysozyme maximum binding capacity was 84 +/- 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation. After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7. 0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer. Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min-1 as shown by the superimposition of the corresponding breakthrough curves. A cartridge assembled with 21 fibers showed a dynamic-to-static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

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.

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 of a lipase from Acinetobacter calcoaceticus AAC323-1 by hydrophobic-interaction methods.

The performance of hydrophobic-interaction chromatography (HIC) for the purification of Acinetobacter calcoaceticus AAC323-1 lipase was compared with that of various aqueous two-phase systems. While a 42% lipase yield with a purification factor of 140 could be recovered by HIC, higher yields were achieved by using aqueous two-phase systems, either those formed by poly(ethylene glycol) and dextran or those based upon the use of a detergent. Triton X-114-based aqueous two-phase partition showed the best performance, with a yield of 81% and a purification factor of 68. Further detergent removal was easily achieved with an adsorbent, with no significant decrease in yields. Owing to its simplicity, the method should be easy to scale-up.

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.

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.

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.

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.