Immobilization of cyanobacteria and microalgae on polyethylenimine-based sorbents

Immobilization of phototrophic microogranisms: microalgae (MA) and cyanobacteria (CB) on polyethylenimine (PEI)-based sorbents was studied. For this purpose, 3 insoluble porous polymeric materials were synthesized by cross-linking of PEI with epichlorohydrine and immobilization of PEI on the surface of styrene–divinylbenzene copolymer. The sorbent on the basis of cross-linked PEI was also alkylated with hexadecyl bromide to achieve hydrophobicity of its surface. The analysis of kinetics and efficiency of immobilization assessed for the model MA and CB cultures revealed the significant difference in the sorption activity of different types of sorbents depending on their synthesis procedure, chemical composition and hydrophilic-hydrophobic properties of polymeric surface. The hydrophobic sorbent obtained by immobilization of PEI on the surface of styrene–divinylbenzene copolymer characterized by very low sorption activity towards CB and MA cells. The highest immobilization efficiency of phototrophic cells was achieved for the hydrophilic sorbent on the basis of PEI cross-linked with epichlorohydrine, which provided the attachment of 50–70% of cells during 3 h of incubation. The hydrophobic sorbent based on alkylated cross-linked PEI effectively immobilized CB cells, while the colonization of the polymer surface by MA cells was very scarce. The noticed effect is explained by difference in prokaryotic (CB) and eukaryotic (MA) types of surface structures organization. Assessment of photosynthetic activity of immobilized MA cells by pulse-modulated fluorometry showed that hydrophobic sorbents had no toxic effect on the cells, while toxicity of hydrophilic cross-linked PEI-based sorbent was observed only after long-term cultivation ofphototrophic cells with this sorbent.     

The use of underloaded C(18) solid-phase extraction plates increases reproducibility of analysis of tryptic peptides from unfractionated human plasma

Bottom-up proteomics requires the digestion of proteins into peptides by processes that use salts for denaturing and buffering purposes. These salts need to be removed prior to mass spectrometry analysis to reduce ion suppression; solid-phase extraction (SPE) is a commonly used strategy. There are many commercially available SPE sorbent types and sizes, which are generally provided with manufacturer recommendations for use, including protein loading capacity. We found that these general suggestions were often not ideal, and our data suggest that context-specific evaluation of sorbent type and amount can improve reproducibility. Specifically, the universal Oasis HLB sorbent provided better retention of the more hydrophilic peptides than the traditional C(18) reversed-phase SPE, but it did so at the expense of an increased loss of the more hydrophobic peptides. We found that increasing the amount of the C(18) sorbent beyond the manufacturer's guidelines decreased breakthrough (i.e., increased retention) of 12 hydrophilic, identifiable peptides without loss of hydrophobic peptides. This procedure was robust in a 96-well plate format.     

Study of the mechanism of interaction of antibody (IgG) on two mixed mode sorbents

Purification of target proteins from a crude biological mixture containing proteins, peptides and other biomolecules is the chromatographic challenge. Mixed mode chromatography offers additional selectivities to improve the overall productivity of commercial bioprocesses with novel chromatographic sorbents being introduced to overcome the problem. HEA HyperCel? (n-hexyl amine) and PPA HyperCel? (phenyl propyl amine) are industry scalable mixed mode chromatography sorbents where both hydrophobic and electrostatic interactions are predominant. Our study focuses on understanding the underlying mechanism of interaction of protein with the sorbent. Parameters like buffer conditions, pH and temperature were tuned to study the adsorption and desorption conditions of the protein. Dynamic binding capacity of HEA HyperCel? and PPA HyperCel? sorbents was studied with human IgG as a model protein. Our study shows that, in HEA the interaction of IgG to the sorbent is predominantly hydrophobic as the binding is enhanced (50–60 mg/ml of sorbent) by presence of salt in buffer and increase in temperature. Binding capacity of PPA is 50–60 mg/ml of sorbent irrespective of temperature effect and/or the presence of salt. The chromatographic experiments show that the interaction could be hydrophobic or ionic or some charge transfer mechanism depending upon the buffer conditions.     

Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression

The secondary structures of proteins (alpha-helical, beta-sheet, beta-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r(2) values of 0.9974, 0.9864, 0.9924, and 0.9743 for alpha-helical, beta-sheet, beta-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r(2) values (r(2) are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 895-905, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Lyotropic salt effects in hydrophobic chromatography.

Pure hydrophobic chromatography can be observed with agarose gels containing caprylyl hydrazide. These nonionic gels show increased avidity in protein adsorption with higher content of caprylyl groups. Lyotropic salt effects can be used to control chromatographic behavior of proteins. Salting-out agents enhance binding of protein to sorbent, while salting-in agents diminish this binding. Based on these findings, the hydrophobic separation of β-lactoglobulin, ovalbumin, and bovine serum albumin is demonstrated.     

Chromatography of carboxylic proteinases on sorbents containing the 2,4-dinitrophenyl group. Role of ionic interactions]

The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described.     

The influence of modified polymeric adsorbents on blood morphologic elements and clotting factors

The changes occurring in human blood after contact with hydrophobic sorbent Vinylsorb S and with ionosorbents Vinylsorb SS were studied. The investigations were carried out using static method at room temperature. The time of contact was 2 h. It was found that the effect of all sorbents upon the morphological elements of blood was small, only platelets were sorbed to a significant extent. The effect of sorbents upon the clotting proteins was also small and plasma hemostasis should not be affected. The sorbents containing sulphonic groups significantly activate kallikreinogenesis and may intensify shock symptoms.     

Equilibria for the adsorption of antibiotics onto neutral polymeric sorbents: Experimental and modeling studies

The objective of this work was to study the equilibria for adsorption of three antibiotics (penicillin V, tetracycline, and cephalosporin C) from water onto commercially available neutral polymeric sorbents. The pH was observed to be an important factor in adsorption as our results suggest that the neutral forms of penicillin V and cephalosporin C are preferentially adsorbed onto the neutral sorbents. Also, sorbent surface chemistry was observed to be important for adsorption, as the antibiotics adsorbed more favorably (both in terms of affinities and enthalpies) onto the aromatic sorbent as compared to the aliphatic ester sorbent. In addition to these thermodynamic measurements, molecular modeling studies and Monte Carlo simulations suggest that adsorption onto aromatic sorbents may involve specific interactions between the planar regions of the antibiotic molecules and the phenyl rings of the aromatic sorbent. The interaction energies predicted from Monte Carlo simulations were observed to provide qualitative agreement with experimentally determined adsorption affinities. (c) 1995 John Wiley & Sons, Inc.     

Optimization of the purification of soil and water objects from oil using biosorbents

Oil biosorbents (patents 2299181, 2318736) were obtained using immobilization of oil-oxidizing microorganisms into the hydrophobic sorbent Sorbonaft, which is manufactured using special technology at the Press-Torf Company. Two associations of aboriginal hydrocarbon-oxidizing microorganisms were used for this purpose: a fungal association and a bacterial and yeast association. The application of biosorbents resulted in a substantial acceleration of the process of purification from oil. The decrease in the amount of oil in the water and soil during 1 month was 30–44% in the variants with the products, against 5% in the control.     

Affinity chromatography of aminopeptidases

The recent data are generalized concerning a series of synthetic oligopeptides which are competitive inhibitors of aminopeptidases of animal, plant and microbic origin. A method for biospecific chromatography of these enzymes is developed, using as ligands such inhibitors as diazo derivatives of p-aminophenyl-, chloromethyl- and methylketones of L-amino acids and peptides, amino acids, aliphatic acid amides. It is established that the most effective inhibitors of aminopeptidases contain L-amino group in the uncharged form in the N-end position, hydrophobic lateral chain of L-configuration and a carbonyl group analogous to position of these groups in the substrate. Methods for synthesis of certain peptides are developed with respect to the above requirements. It is shown that peptides with a space-inaccessible peptide link and antibiotics are often used as ligands for affinity chromatography of aminopeptidases. At present a nonspecific (ion-exchange, hydrophobic) interaction of sorbent and aminopeptidases is observed, which necessitates to increase the specificity at the stage of the enzyme desorption in the further studies.     

Recombinant human insulin: X. Simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing of Cys residues. The inclusion bodies were dissolved in 8 M urea with addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Albumin mRNA from rat liver cells. Analysis of immunoadsorption of individual polyribosomes]

The analysis of albumin polyribosomes immunoadsorption is carried out using "sandwich" immunoadsorbents prepared on the basis of two aminobenzylcelluloses: commercial (paraaminobenzylcellulose) and synthesised (methaaminobenzyloxymethylcellulose). A method is worked out which is good for the estimation of the adaptibility of different aminobenzylcellulose preparations as an insoluble basis for the immunoadsorbent. Major properties of the "sandwich" sorbent (the accessible capacity and specificity) and the percent of isolated individual polyribosomes are found to be interrelated and determined by conditions of the immunoadsorption reaction. The increase of polyribosomes and sorbent concentrations and their ratio in the incubation medium results in the increase of the sorbent accessible capacity and the decrease in the inspecific adsorption but at the same time the percent of adsorbed polyribosomes decrease too. The "excess" of adsorbent with respect to polyribosomes, participating in the binding reaction, is necessary for the quantitative isolation of individual polyribosomes.     

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Features of protein adsorption by silicopolymethylsiloxanes

The adsorption of tripsin and albumin on silicapolymetylsilocsan (SG-PMS) and its modified forms by cooper (II)--(SG-PMS (Cu)--from water-salt solutions were studied. It was determined difference in peculiarities of proteins sorption depending on its amino acids composition, chemistry of sorbent surface and acidity of medium. It was showed, that modified by cooper (II) sorbent have high affinity to tripsin than albumin in studied solutions. Influence of bearer modification degree on albumin immobilisation and interaction character of SG-PMS (Cu) active centres with function groups of enzyme and albumin have been showed.     

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.     

Microcolumn exclusion HPLC of globular proteins with refractive index detection

A method of determination of molecular masses of globular proteins based on microcolumn size-exclusion HPLC on modified silica sorbent (TSK-GEL 3000 SW) with refractometric detection has been developed. Molecular masses in the range 10 to 200 kD were determined with the accuracy of +/- 3%.     

Inclusion complex-based solid-phase extraction of steroidal compounds with entrapped beta-cyclodextrin polymer

Although the hydrophobic interaction-based solid-phase extraction (SPE) has been widely used, the extraction yields of steroids including androgens, estrogens, and corticoids were slightly different along with the physical and chemical properties of each molecule. A new SPE technique based on the formation of an inclusion complex with beta-cyclodextrin (betaCD) has been achieved for comprehensive sample purification in mass spectrometric analysis of 45 endogenous or synthetic androgens, 11 endogenous estrogens, and 21 corticoids. A copolymer of betaCD with epichlorohydrin was prepared by a cross-linking reaction followed by entrapment with 0.3M CaCl(2) to yield an improved SPE sorbent and the hydrolyzed urine samples were applied for purification. Steroidal compounds tested on the entrapped betaCD polymer were extracted with tetrahydrofuran and the overall recoveries ranged from 82% to 112% for 77 steroids in urine. Especially, the hydroxylated estrogens showed an excellent binding capacity (96-116% recovery) to betaCD through hydrogen bonding between their phenolic hydroxyl and exterior hydroxyl groups. A comparison between SPE methods with betaCD and Oasis HLB as a conventional cartridge showed that the extraction efficiency of polar steroids was significantly increased in the betaCD experiment, which has no connection with different polarity of steroid molecules. Due to its multi-functional mechanism derived from molecular inclusion and chemical interactions, this new SPE sorbent resulted in better selectivity and extraction efficiency than that obtained using the conventionally used hydrophobicity-based SPE method.     

A strategy for high-throughput screening of ligands suitable for molecular imprinting of proteins

For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates. In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin - a protein of the protease family - was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach. The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin.     

Metal proteases from Bac. subtilis]

Metal and serine proteases were separated on the biospecific sorbent. Two different, homogeneous metal proteases were obtained by rechromatography of the metal protease. Activation energies, heat stability, molecular weights, influence of inhibitors, the dependence of activity on pH and temperature were determined. Properties of two metal proteases were compared with those of literary analogs.     

Ion Exclusion Chromatography: Parameters Influencing Retention

Ion Exclusion Chromatography (IEC) finds application in the separation of a wide range of small, neutral or partially ionized molecules. In IEC, the strong as well as weak electrolytes are eluted unseparated, the first at the beginning and the latter at the end of the elution. The retention volumes of the remaining electrolytes are found to be proportional to their dissociation constant values. The dead and inner volumes of the chromatographic column can be determined from the observed dependence of retention volumes on dissociation constant values. The retention mechanism is described by the analytical equations and by the results obtained from the computer simulation of the column performance (using global thermodynamic and chromatographic equations or the Craig method). The mixed retention mechanism involving hydrophobic adsorption and screening effect is observed for weak electrolytes and aromatic compounds. Aromatic compounds are found to be retained almost solely by a reverse-phase mechanism involving interaction of the solute with the unfunctionalized regions of the stationary phase. The purpose of this paper is to survey the field. Using theoretical and experimental approaches, I show how different parameters can influence ion-exclusion solute retention. Although this retention is affected by the physicochemical parameters of the sorbent, stationary and mobile phases especially, this study primarily deals with the structural solute parameters, stationary phase form and column temperature, that have had little or no discussion in literature.