Combined protein A imprinting and cryogelation for production of spherical affinity material

Cryogels have been demonstrated to be efficient when applied for protein isolation. Owing to their macroporous structure, cryogels can also be used for treating particle‐containing material, e.g. cell homogenates. Another challenging development in protein purification technology is the use of molecularly imprinted polymers (MIPs). These MIPs are robust and can be used repeatedly. The paper presents a new technology that combine the formation of cryogel beads concomitantly with making imprints of a protein. Protein A was chosen as the print molecule which was also be the target in the purification step. The present paper describes a new method to produce protein‐imprinted cryogel beads. The protein‐imprinted material was characterized and the separation properties were evaluated with regard to both the target protein and whole cells with target protein exposed on the cell surface. The maximum protein A adsorption was 18.1 mg/g of wet cryogel beads. The selectivity coefficient of protein A‐imprinted cryogel beads for protein A was 5.44 and 12.56 times greater than for the Fc fragment of IgG and protein G, respectively.     

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.     

Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

Surface-modified membranes as a matrix for protein purification.

Recently introduced membrane-based chromatographic supports for protein separation are available either with a coupled ligand, e.g., protein A, protein G or ion-exchange groups, or as activated matrices for coupling a desired ligand. The coupling conditions for protein A and immunoglobulin G to an epoxy-activated membrane were determined. The performance of the prepared affinity membranes was investigated using pure rabbit immunoglobulin G and protein A as a model system. For practical application monoclonal antibodies from cell culture supernatant were purified with a prepared protein A membrane and for comparison with a sulphonic acid ion exchange membrane.     

Purification of streptococcal protein G expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin

A one-step HPLC method was developed for the purification of protein G, a cell wall molecule from group C and G streptococci with immunoglobulin G- and albumin-binding properties. Lysed Escherichia coli bacteria infected with lambda-phages containing the protein G gene from group G streptococci were used as a starting material for the preparations. The lysate was applied to a column with immobilized human immunoglobulin G or human serum albumin. Protein G was selectively bound and eluted at pH 2.0. A 750-fold purification was achieved. Sodium dodecylsulfate + polyacrylamide gel electrophoresis showed that the highly purified protein G consisted of three sets of doublets with the apparent molecular weight of 64 and 67, 56 and 58, and 45 and 47 kilodaltons, respectively. A specific method for quantitation of small amounts of protein G was developed and used for specific tracing of the protein after the affinity chromatography. Goat polyclonal antibodies were bound to an antigen coated to the plastic walls of microtiter plates, causing the Fc-region of the immunoglobulins to be directed outwards. Unknown samples of protein G were then allowed to compete with radio-iodinated protein G (solid phase radioassay) or protein G coupled to alkaline phosphatase (enzyme linked sorbent assay) for the Fc-regions.     

Effect of the conserved oligosaccharides of recombinant monoclonal antibodies on the separation by protein A and protein G chromatography

Glycosylation of the conserved asparagine residue in CH2 domains of IgG molecules is an important post-translational modification. The presence of oligosaccharides is critical for structure, stability and biological function of IgG antibodies. Effect of the glycosylation states of recombinant monoclonal antibodies on protein A and protein G chromatography was evaluated. Antibodies lacking oligosaccharides eluted later from protein A and earlier from protein G columns than antibodies with oligosaccharides using a gradient of decreasing pH. Interestingly, different types of oligosaccharides also affected the elution of the antibodies. Antibodies with high mannose type oligosaccharides were enriched in later eluting fractions from protein A and earlier eluting fractions from protein G. While antibodies with more mature oligosaccharides, such as core fucosylated biantennary complex oligosaccharides with zero (Gal 0), one (Gal 1) or two (Gal 2) terminal galactoses, were enriched in earlier eluting fractions from protein A and in the later eluting fractions from protein G. However, analysis by enzyme-linked immunosorbent assay (ELISA) revealed that antibody binding affinity to protein A and protein G was not affected by the absence or presence of oligosaccharides. It was thus concluded that the elution difference of antibodies with or without oligosaccharides and antibodies with different types of oligosaccharides were due to differential structural changes around the CH2–CH3 domain interface under the low pH conditions used for protein A and protein G chromatography.     

Metal Affinity-Based Purification of a Red Fluorescent Protein

A red fluorescent protein, DsRed, which emits fluorescence in the red region of the spectrum has become a popular alternative to green fluorescent protein as a label in biochemical and bioanalytical applications. In this study, we have developed a simple purification method for DsRed variants utilizing their inherent copper binding property. A purification procedure was developed and optimized using immobilized copper ions yielding a single strong band corresponding to purified DsRed proteins on the SDS-PAGE gel. A purification efficiency of higher than 95% was achieved. A spectral analysis and copper binding study was performed to verify activity of the purified proteins. The development of this method allows DsRed to play a dual role as a fluorescent reporter protein and as a purification affinity tag for a target protein. This simpler approach of purification should expand the utility of DsRed.     

Synthesis and evaluation of bioorthogonal pantetheine analogues for in vivo protein modification

In vivo carrier protein tagging has recently become an attractive target for the site-specific modification of fusion systems and new approaches to natural product proteomics. A detailed study of pantetheine analogues was performed in order to identify suitable partners for covalent protein labeling inside living cells. A rapid synthesis of pantothenamide analogues was developed and used to produce a panel which was evaluated for in vitro and in vivo protein labeling. Kinetic comparisons allowed the construction of a structure-activity relationship to pinpoint the linker, dye, and bioorthogonal reporter of choice for carrier protein labeling. Finally bioorthogonal pantetheine analogues were shown to target carrier proteins with high specificity in vivo and undergo chemoselective ligation to reporters in crude cell lysate. The methods demonstrated here allow carrier proteins to be visualized and isolated for the first time without the need for antibody techniques and set the stage for the future use of carrier protein fusions in chemical biology.     

Per Aqueous Liquid Chromatography (PALC) as a Simple Method for Native Separation of Protein A

Staphylococcal protein A (protein A) is an important protein frequently used in research studies within the fields of biomedicine and biotechnology. Due to some limitations in available protein purification methods which can hold the native structure of the protein A without changing the folding or adding histidine to structure of this protein, its separation in the native form is difficult. In this study, a new cost-effective and powerful technique was introduced for separation of the full-length and truncated forms of recombinant protein A, without any alteration in their 3D structures. Per aqueous liquid chromatography with bare silica gel stationary phase and water:acetonitrile as the mobile phase was proved to be an attractive choice among the range of separation methods. Similar to hydrophilic liquid chromatography, this method employs high percentage of water in mobile phase. The effects of mobile phase composition, pH, and salt concentration on the retention behavior of protein A on bare silica gel stationary phases were investigated. In this method, applying high amounts of aqueous solvent accompanied by a minimum percentage of organic solvent could successfully separate protein A with preservation of folding, and any affinity-tagged group such as histidine has not occurred on its structure. Purity of the fractions obtained by the proposed method was confirmed using SDS-PAGE, western blotting, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. According to the results of ELISA, separated proteins retained their ability of binding to antibody.     

Use of microfiltration as first step in recovery of protein a from fermentation broth

The flux and transmission of protein A during microfiltration have been studied. We studied the performance of two commercial membranes: one made of nylon (Pall Ultipore Nylon₆₆, 0.2 μm) and one of polyether sulfone (Pall Omega, 0.16 μm). The Nylon₆₆ membrane had by far the best transmission of protein A although a previous study showed that bovine serum albumin (BSA), often used to characterize membranes, had much better transmission through the Omega membrane. The membrane manufacturer also states that the Omega membrane is the best membrane for this kind of application because it is a low-protein-binding membrane. The lower transmission of the Omega membrane for protein A was assumed to be owing to its smaller pores and higher charge density in combination with the larger Stokes radius for protein A. When the pH was lowered, the Nylon₆₆ membrane still had the higher transmission. It can thus be concluded that a membrane that is found suitable for the recovery process of one protein is not always the best choice for the recovery process for other proteins even though the membrane is low protein binding.     

The effect of feed composition on the behavior of chemically selective displacement systems

In this paper we examine whether adding a more retained protein to the feed will mitigate displacer–protein interactions in the column, thus affecting the displacement modality that occurs (chemically selective vs. traditional displacement chromatography). STD-NMR experiments were carried out to probe displacer–protein interactions for the chemically selective displacer chloroquine diphosphate and the results indicated that this displacer only had measurable interactions with the protein α-chymotrypsinogen A. For a two component feed mixture containing ribonuclease A and α-chymotrypsinogen A, the separation resulted in the displacement of ribonuclease A, with the more hydrophobic α-chymotrypsinogen A remaining on the column. On the other hand, when the experiment was repeated with cytochrome c added to the feed, all three feed proteins were displaced. Column simulations indicated that the combination of sample self-displacement occurring during the introduction of the feed, along with the dynamics of the initial displacement process at the column inlet was responsible for this behavior. These results indicate that for this class of hydrophobic-based selective displacers, in order for the protein to be selectively retained, the protein should be the most strongly retained feed component.     

Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Setup and detector performance

A lamp‐based fluorescence detection (Flu) system for CE was extended with a wavelength‐resolved (WR) detector to allow recording of full protein emission spectra. WRFlu was achieved using a fluorescence cell that employs optical fibres to lead excitation light from a Xe‐Hg lamp to the capillary window and protein fluorescence emission to a spectrograph equipped with a CCD. A 280 nm band pass filter etc. together with a 300 nm short pass cut‐off filter was used for excitation. A capillary cartridge was modified to hold the detection cell in a commercial CE instrument enabling WRFlu in routine CE. The performance of the WRFlu detection was evaluated and optimised using lysozyme as model protein. Based on reference spectral data, a signal‐intensity adjustment was introduced to correct for transmission losses in the detector optics that occurred for lower protein emission wavelengths. CE‐WRFlu of lysozyme was performed using BGEs of 50 mM sodium phosphate (pH 6.5 or 3.0) and a charged‐polymer coated capillary. Using the 3‐D data set, signal averaging over time and emission‐wavelength intervals was carried out to improve the S/N of emission spectra and electropherograms. The detection limit for lysozyme was 21 nM, providing sufficient sensitivity to obtain spectral information on protein impurities.     

Characterization and Activity Determination of the Human Protein Phosphatase 2A Catalytic Subunit α Expressed in Insect Larvae

Protein phosphatase 2A is the major enzyme that dephosphorylates the serine/threonine residues of proteins in the cytoplasm of animal cells. This phosphatase is most strongly inhibited by okadaic acid. Besides okadaic acid, several other toxins and antibiotics have been shown to inhibit protein phosphatase 2A, including microsystin-LR, calyculin-A, tautomycib, nodularin, cantharidine, and fostriecin. This makes protein phosphatase 2A a valuable tool for detecting and assaying these toxins. High-scale production of active protein phosphatase 2A requires processing kilograms of animal tissue and involves several chromatographic steps. To avoid this, in this work we report the recombinant expression and characterization of the active catalytic subunit ?? of the protein phosphatase 2A in Trichoplusia ni insect larvae. Larvae were infected with baculovirus carrying the coding sequence for the catalytic subunit ?? of protein phosphatase 2A under the control of the polyhedrin promoter and containing a poly-His tag in the carboxyl end. The catalytic subunit was identified in the infected larvae extracts, and it was calculated to be present at 250???g per gram of infected larvae, by western blot. Affinity chromatography was used for protein purification. Protein purity was determined by western blot. The activity of the enzyme, determined by the p-nitrophenyl phosphate method, was 94???mol/min/mg of purified protein. The catalytic subunit was further characterized by inhibition with okadaic acid and dinophysis toxin 2. The results presented in this work show that this method allows the production of large quantities of the active enzyme cost-effectively. Also, the enzyme activity was stable up to 2?months at ?20?°C.     

Isoelectric focusing in cyclic olefin copolymer microfluidic channels coated by polyacrylamide using a UV photografting method

As an alternative material to glass or silicon, microfluidic devices made from a cyclic olefin copolymer (COC) were fabricated. This material is of interest because of the relative ease of fabrication, low costs, and solvent resistance. However, as a result of the strong hydrophobic interactions normally present, COC surfaces are not suitable for protein separations. To reduce the protein adsorption and make COC suitable for protein separations, UV-initiated grafting of polyacrylamide was used to coat the surface of COC devices. The change in surface properties caused by different graft times was studied. The surface hydrophilicity and electroosmotic mobility were characterized by contact angle and electroosmosis measurements. Isoelectric focusing was performed to test protein separations in polyacrylamide-coated COC microchannels. A single protein, carbonic anhydrase, was used to analyze the focusing effects and peak capacities in uncoated and polyacrylamide-coated COC devices. Peak capacities ranging from 75 to 190 were achieved with a polyacrylamide-coated surface. A mixture of two proteins, conalbumin labeled with Alexa Fluor 488 and beta-lactoglobulin A labeled with Alexa Fluor 546, was used to test protein separations. Linear and rapid separation of proteins was achieved in the polyacrylamide-coated COC microfluidic device.     

液相色谱-质谱联用分析流感病毒感染引起宿主蛋白类泛素化修饰

干扰素刺激基因15编码蛋白质(Interferon stimulated gene 15 kDa protein, ISG15)是最早被鉴定的类泛素分子蛋白质,在病毒感染和免疫调节等方面具有重要作用。本研究利用免疫沉淀技术将被类泛素 ISG15修饰的蛋白富集纯化,采用液相色谱-质谱联用技术对流感病毒感染 A549宿主细胞过程中产生的类泛素 ISG15修饰蛋白进行了分析。实验结果表明,在流感病毒感染的实验组 A549细胞中,鉴定到了22种来源于宿主细胞的ISG15修饰的蛋白,包括类泛素蛋白 ISG15、细胞周期蛋白-T1、热休克蛋白71、钙调素结合蛋白、真核翻译起始因子等,以及1种来源于流感病毒的非结构蛋白 NS1。在鉴定的22种宿主蛋白中,有6种蛋白在未感染病毒的对照组 A549细胞中也得到鉴定,包括膜联蛋白 A1、果糖二磷酸醛缩酶 A、线粒体三磷酸腺苷合成酶亚基 g、烯醇化酶、肌动蛋白、微管蛋白。生物信息学分析表明,流感病毒感染引起的 ISG15修饰的宿主蛋白分别归属于9个不同的蛋白分类,包括细胞骨架蛋白、分子伴侣蛋白、酶调节剂、核酸结合蛋白、激酶类、转移酶类、转录因子、氧化还原酶类以及结构蛋白。本研究为大规模分析鉴定 ISG15修饰蛋白提供了一种特异、有效的研究方法。     

The application of high resolution two-dimensional polyacrylamide gel electrophoresis to the identification and purification of a protein, NG8.4, present in Neisseria gonorrhoea and the subsequent development of a radioimmunoassay

A soluble protein of unknown function was shown, by two-dimensional polyacrylamide gel electrophoresis, to be present in Neisseria gonorrhoeae at significantly higher concentrations than in the other related bacteria tested. The data indicate the possibility of this protein being specific to Neisseria gonorrhoeae. This protein was designated as NG 8.4 and purified. A radioimmunoassay was developed for the measurement of this protein and subsequently used to determine the degree of cross reaction exhibited by a number of species of bacteria. Of the bacteria tested only those of the genus Neisseria gave a significant reaction in the assay.     

Gellan beads as a transparent media for protein immobilization and affinity capture

Gellan gum beads are presented as a novel substrate for protein immobilization and immobilized protein activity measurements. The optical transparency of the gellan beads down to 200 nm provides a method for direct quantitation of the amount of protein immobilized onto the beads. The ability to utilize these beads in a non-aqueous activation step allowed for a fourfold increase in the amount of protein immobilized, and this method was used to immobilize Protein A onto gellan beads at a final yield of 1.42+/-0.07 mg of Protein A/g of beads. The optical transparency also allowed for detection of the activity of the immobilized Protein A simply by measuring the absorbance of the beads following capture of rabbit IgG. This activity measurement method was compared with a traditional method utilizing the amount of protein remaining in solution after the IgG capture step. The traditional method yielded an activity measurement of 10.9+/-0.2 mg IgG/mg of Protein A, while the absorbance method showed an activity of only 7.5+/-0.3 mg IgG/mg of Protein A. The difference can be explained by the more direct measurement used in the absorbance method. The optical transparency of the beads was also evaluated in a fluorescence based IgG capture experiment, showing that detection of fluorescent IgG captured on the beads was possible with no interference from the beads.     

Flow Injection Fluoroimmunoassay for Human Transferrin Using a Protein a Immunoreactor

Abstract

A protein A immunoreactor (immobilized protein A) incorporating flow injection technique was used for on-line fluoroimmunoassay of human transferrin. Antibody immobilized on protein A and antibody-antigen complex formation took place in phosphate buffered saline (PBS, PH 7.4). After washing off excess lucifer yellow VS labelled human transferrin, the antibody-antigen complex was eluted with acid buffer and detected. Experimental variables have been studied and the method has been used to determine the transferrin contents in human serum.     

Comparative Assessment of Oriented Antibody Immobilization on Surface Plasmon Resonance Biosensing

Protein A and protein G are extremely useful molecules for the immobilization of antibodies. However, there are limited comparative reports available to evaluate their immobilization performance for use as biosensors. In this study, a comparative analysis was made of approaches that use protein A and protein G for avian leukosis virus detection. The antibody‐protein binding affinities were determined using surface plasmon resonance (SPR) analysis. The immobilization efficiency was obtained by calculating the number of the protein molecular binding sites. The positive influence of sensor response on antigen detection indicates that the amount of immobilized antibody plays a major role in the extent of immobilization. Moreover, the biosensors constructed using both proteins were found to be regenerative. The SPR results from this study suggest that the surfaces of protein G provide a better equilibrium constant and binding efficacy for immobilized antibodies, resulting in enhanced antigen detection.     

Affinity purification of antibodies using immobilized FB domain of protein A.

A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.