Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix((R)) Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel((R)) A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 muM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C). (c) 1995 John Wiley & Sons, Inc.     

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab‐binding domain of Streptococcal Protein G (SpG‐domain III). The ligand (A2C7I1) was synthesized by the four‐component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer‐aided analysis suggests a putative binding site on the C_H1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization–mass spectrometry and ¹H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml^?1 and a dissociation constant of 5.34 × 10^?5 M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

Corn offers advantages as a transgenic host for producing recombinant proteins required at large volumes (1,000's of tons per year) and low cost (less than US$50/kg) by generating them as co‐products of biorefining. We describe the purification and characterization of a corn grain‐derived mammalian structural protein having such market characteristics: a full length recombinant collagen type I alpha 1 (rCIα1) chain. Material properties of interest are gelation behavior, which would depend on as yet unverified ability of corn to carry out post‐translational prolyl hydroxylation and formation of triple helical conformation. The starting material was grain where the expression of rCIα1 had been directed by an embryo‐specific promoter. Purification consisted of extraction at low pH followed by membrane and chromatographic steps to isolate rCIα1 for characterization. The amino acid composition and immunoreactivity of CIα1 was similar to that of an analogous native human CIα1 and to rCIα1 produced by the yeast Pichia pastoris. Tandem mass spectrometry confirmed the primary sequence of the corn‐derived rCIα1 with 46% coverage. Fragments of the rCIα1chains were also observed, possibly caused by endogenous plant proteases. The corn‐derived rCIα1 had a low level of prolyl hydroxylation (～1% versus 11%) relative to animal‐derived CIα1 and folded into its characteristic triple‐helical structure as indicated by its resistance to pepsin digestion below its melting temperature of 26^oC. The 29 amino acid foldon fused to the C‐terminus to initiate triple helix formation was not cleaved from the rCIα1chains, but could be removed by pepsin treatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

A novel and conserved pocket of human kappa-Fab fragments: design, synthesis, and verification of directed affinity ligands

Antibodies of type IgG may be divided into two classes, called lambda or kappa, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG kappa-Fab, which is not present in the lambda type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group-containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of kappa-Fab fragments with different variable regions, whereas a sample of monoclonal lambda-Fab fragments was not retained under similar chromatographic conditions.     

Design,synthesis and characterisation of affinity ligands for glycoproteins

The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar‐binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine‐based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd.     

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.     

Relative stability of G‐quadruplex structures: Interactions between the human Bcl2 promoter region and derivatives of carbazole and diphenylamine

The bcl2 promoter region forms a G‐quadruplex structure, which is a crucial target for anticancer drug development. In this study, we provide theoretical predictions of the stability of different G‐quadruplex folds of the 23‐mer bcl2 promoter region and G‐quadruplex ligand. We take into account the whole G‐quadruplex structure, including bound‐cations and solvent effects, in order to compute the ligand binding free energy using molecular dynamics simulation. Two series of the carbazole and diphenylamine derivatives are used to screen for the most potent drug in terms of stabilization. The energy analysis identifies the predominant energy components affecting the stability of the various different G‐quadruplex folds. The energy associated with the stability of the G‐quadruplex‐K⁺ structures obtained displays good correlation with experimental T_m measurements. We found that loop orientation has an intrinsic influence on G‐quadruplex stability and that the basket structure is the most stable. Furthermore, parallel loops are the most effective drug binding site. Our studies also demonstrate that rigidity and planarity are the key structural elements of a drug that stabilizes the G‐quadruplex structure. BMVC‐4 is the most potential G‐quadruplex ligand. This approach demonstrates significant promise and should benefit drug design. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1038–1050, 2014.     

Evaluation of new affinity chromatography resins for polyclonal,oligoclonal and monoclonal antibody pharmaceuticals

The performance of MabSelect SuRe and IgSelect affinity chromatography resins designed for process-scale purification of antibodies was investigated. Various antibodies (4 human monoclonal, 1 human polyclonal and 1 bovine polyclonal antibody and 1 Fc-fusion protein) were used to evaluate the elution pH and dynamic binding capacity of the resins. The elution pH for each human antibody was similar on MabSelect SuRe and IgSelect (pH 3.5–3.8). No significant differences in dynamic binding capacity were observed among human antibodies on MabSelect SuRe (∼20–40 mg/mL resin) and IgSelect (∼10–30 mg/mL resin). The binding capacity order for the human antibodies was the same on MabSelect SuRe and IgSelect. Using a linear pH gradient, both resins were able to partially separate monomeric and aggregated forms of the antibodies. The results indicate that these new affinity resins are powerful tools for the purification of human polyclonal antibodies from transgenic animals and oligoclonal antibodies from CHO cell cultures.     

Isolation,purification, and identification of the virulence protein VirE2 from <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Bacteria of the genus Agrobacterium are capable of transferring a fragment of their Ti-plasmid T-DNA, in a complex with the proteins VirE2 and VirD2, into the nuclei of plant cells and incorporating it into the chromosome of the host. The mechanisms of T-DNA transportation through the membrane and cytoplasm of the plant cell are unknown. The aim of this work was isolation of the virulence protein VirE2 for studying its role in T-DNA transportation through the membrane and cytoplasm of eukaryotic cells. For VirE2 accumulation, the virE2 gene was cloned into plasmid pQE31. VirE2 was isolated from the cells of E. coli strain XL1-blue, containing the recombinant plasmid pQE31-virE2. The cells were disrupted ultrasonically, and the protein, with six histidine residues at the N-end, was isolated by means of affinity chromatography on a Ni-NTA-superose column. The purified protein was tested by the immunodot method using polyclonal rabbit antibodies and anti-VirE2 miniantibodies. The ability of the recombinant protein VirE2 to bind to single-stranded DNA was judged from the formation of complexes detected by electrophoresis in agarose gel. Thus, we isolated, purified, and partially characterized the Agrobacterium tumefaciens virulence protein VirE2, which is capable of binding to single-stranded T-DNA upon transfer to the plant cell.Translated from Mikrobiologiya, Vol. 74, No. 1, 2005, pp. 92–98.Original Russian Text Copyright © 2005 by Volokhina, Sazonova, Velikov, Chumakov.     

Design,synthesis and application of chiral tetraoxacalix[2]arene[2]triazine‐based organocatalysts in asymmetric Michael addition reactions

A novel type of oxacalix[2]arene[2]triazine‐based organocatalysts for asymmetric Michael reactions are reported for the first time. Chiral subunits were attached to the heteroatom‐bridged calixaromatic platform by a reaction of (R)‐ and (S)‐1‐aminotetraline with tetraoxacalix[2]arene[2]triazine in both enantiomeric forms. To evaluate the catalytic efficiency of the novel organocatalysts, isobutyraldehyde reacted with various substituted and unsubstituted aromatic trans‐β‐nitrostyrenes in tetrahydrofuran (THF), leading to Michael adducts in excellent yields and enantioselectivites (up to 97% yield and 99% ee).     

Multi‐steps green process for synthesis of six‐membered functional cyclic carbonate from trimethylolpropane by lipase catalyzed methacrylation and carbonation,and thermal cyclization

A highly functionalized six‐membered cyclic carbonate, methacrylated trimethylolpropane (TMP) cyclic carbonate, which can be used as a potential monomer for bisphenol‐free polycarbonates and isocyanate‐free polyurethanes, was synthesized by two steps transesterifications catalyzed by immobilized Candida antarctica lipase B, Novozym^®435 (N435) followed by thermal cyclization. TMP was functionalized as 70 to 80% selectivity of mono‐methacrylate with 70% conversion was achieved, and the reaction rate was evaluated using various acyl donors such as methacrylic acid, methacrylate‐methyl ester, ‐ethyl ester, and ‐vinyl ester. As a new observation, the fastest rate obtained was for the transesterfication reaction using methacrylate methyl ester. Byproducts resulted from leaving groups were adsorbed on the molecular sieves (4Å) to minimize the effect of leaving group on the equilibrium. The difference of reaction rate was explained by molecular dynamic simulations on interactions between carbonyl oxygen and amino acid residues (Thr 40 and Glu 157) in the active site of lipase. Our docking studies revealed that as acyl donor, methyl ester was preferred for the initial conformation of the first tetrahederal intermediate with hydrogen bonding interactions. TMP‐monomethacrylate (TMP‐mMA) cyclic carbonate was obtained in 63% yield (74.1% calculated in 85% conversion) from the lipase‐catalyzed carbonation reaction of TMP‐mMA with dimethylcarbonate, and followed by thermal cyclization of the monocarbonate at 90°C. From the multiple reactions demonstrated in gram scale, TMP‐mMA cyclic carbonate was obtained as a green process without using chlorinated solvent and reagent. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:83–88, 2016     

Structural and functional characterization of the single-chain Fv fragment from a unique HCV E1E2-specific monoclonal antibody

The nucleotide sequence of the unique neutralizing monoclonal antibody D32.10 raised against a conserved conformational epitope shared between E1 and E2 on the serum-derived hepatitis C virus (HCV) envelope was determined. Subsequently, the recombinant single-chain Fv fragment (scFv) was cloned and expressed in Escherichia coli, and its molecular characterization was assessed using multi-angle laser light scattering. The scFv mimicked the antibody in binding to the native serum-derived HCV particles from patients, as well as to envelope E1E2 complexes and E1, E2 glycoproteins carrying the viral epitope. The scFv D32.10 competed with the parental IgG for binding to antigen, and therefore could be a promising candidate for therapeutics and diagnostics.     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display,in silico loop reconstruction and molecular dynamics simulations

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.