Immunoaffinity purification of α-momorcharin from bitter melon seeds (Momordica charantia)

α‐Momorcharin (α‐MMC), a type I ribosome‐inactivating protein (RIP), has shown therapeutic potential such as anti‐tumor and anti‐viral agent. Traditional process of α‐MMC purification from bitter melon seeds was time consuming and low efficient. To take this challenge, we made an affinity matrix by coupling the monoclonal antibody (McAb) with Sepharose 4B. Using this attractive strategy, 196 mg of α‐MMC was obtained from 100 g of bitter melon seeds as the starting material. The yield of the protein was 2.7%. The homogeneity and properties of the protein were assessed by SDS‐PAGE, acidic PAGE, RP‐HPLC and N‐terminal sequence as well as Western blot. Purified α‐MMC showed remarkable inhibition to the melanoma cell line JAR and EMT‐62058. In addition, it also displayed obvious inhibition on hepatitis B virus (HBV). This work provided a simple, rapid and efficient approach for α‐MMC purification from Momordica charantia.     

Construction and Characterization of an Anti‐Prion scFv Fusion Protein Pair for Detection of Prion Protein on Antibody Chip

Abstract

A pair of single chain Fv fragment (scFv) fusion proteins were constructed and characterized. Antibody chips using the pair were designed for sensitive detection of prion protein. Phage displayed antibody library was synthesized by immunizing mice with thioredoxin‐mature bovine prion fusion protein (TrxA‐bPrP^c). After five rounds of panning against recombinant bovine prion protein (rb‐PrP^c) and ELISA test, two positive clones with high affinity to rb‐PrP^c, named Z163 and Z186, were obtained. They were conjugated with a linker‐streptavidin binding protein (SBP) or human IgG1 constant fragment (Fc) to form the scFv fusion protein pair Z186‐L‐SBP/Z163‐Fc. Western blot experiments showed that the scFv fusion pair specifically interacted with the line epitopes of the protease resistant core region bPrP27‐30. Surface plasmon resonance (SPR) sensorgrams revealed that the equilibrium dissociation constants of the interactions with rb‐PrP^c were 3.24×10^?8 M, 8.82×10^?8M, and 8.10×10^?9 M for Z186‐L‐SBP, Z163, and Z163‐Fc, respectively. All binding reactions followed rapid association and slow dissociation kinetics. As a detection pair, Z186‐L‐SBP functioned as a capture probe and was immobilized on the streptavidin coated slides to form reactive layer of the antibody chip, and Z163‐Fc labeled with fluorescence dye Cy3 functioned as a detection probe generating fluorescence signal. The antibody chip could detect existence of rb‐PrP^c with detection limit of 1 pg/ml.     

A fully automated linear polyacrylamide coating and regeneration method for capillary electrophoresis of proteins

Surface modification of the inner capillary wall in CE of proteins is frequently required to alter EOF and to prevent protein adsorption. Manual protocols for such coating techniques are cumbersome. In this paper, an automated covalent linear polyacrylamide coating and regeneration process is described to support long‐term stability of fused‐silica capillaries for protein analysis. The stability of the resulting capillary coatings was evaluated by a large number of separations using a three‐protein test mixture in pH 6 and 3 buffer systems. The results were compared to that obtained with the use of bare fused‐silica capillaries. If necessary, the fully automated capillary coating process was easily applied to regenerate the capillary to extend its useful life‐time.     

Designing and monitoring large-scale processes for biomolecule purification with an automated electrophoresis/staining system

An automated electrophoresis/staining system is advantageous in the design, evaluation and documentation of biomolecule purification processes. Its use from laboratory scale to industrial scale is demonstrated in three major purification processes: (i) superoxide dismutase production (processing 3.51 of a clarified yeast lysate), (ii) staphylococcal enterotoxin B production (processing 440 l of supernatant liquid from a cell culture), and (iii) monoclonal antibody production (processing 143 l of a supernatant liquid from a cell culture). The speed and convenience of the system provide advantages in process monitoring; automated silver staining (60 min) enabled the detection of <0.5 ng μl^?1 contaminating protein.     

Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development

Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western? (or Simon?) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon? can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.     

Characterization of therapeutic antibody fragmentation using automated capillary western blotting as an orthogonal analytical technique

Fragmentation in protein‐based molecules continues to be a challenge during manufacturing and storage, and requires an appropriate control strategy to ensure purity and integrity of the drug product. Electrophoretic and chromatographic methods are commonly used for monitoring the fragments. However, size‐exclusion chromatography often suffers from low resolution of low molecular weight fragments. Electrophoretic methods like CE‐SDS are not compatible with enriching fragments for additional characterization tests such as MS. These limitations may result in inadequate control strategy for monitoring and characterizing fragments for protein‐based molecules. Capillary western blotting was used in this study as an orthogonal method for characterization of fragments in an IgG1 antibody under reduced conditions. To achieve a comprehensive mapping of various fragments generated by thermal stress, capillary western profiles were generated using recognition antibodies for IgG kappa (κ) light chain, Fc, and Fab regions that enabled unambiguous fragment identification. Additionally, three different enzymatic digestion methods (IdeS, PNGase F, and IgdE) were applied coupled with capillary western blotting for clip identifications. Finally, complementary data collected using traditional chromatographic and electrophoretic methods allowed to establish a comparison of analytical profiles with an added benefit of fragment identification offered by capillary western profiling. In addition to various Fc and Fab‐related low molecular weight fragments, a non‐reducible thio‐ether linked 75 kDa HL fragment was also identified.     

猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定

以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,westem blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.     

Isolation of recombinant mycobacterial antigens by an automatic and generally applicable purification method for beta-galactosidase fusion proteins

An automated two-dimensional chromatographic method has been developed for the isolation and concentration of recombinant fusion proteins with beta-galactosidase. The system consists of an immunoaffinity column with anti-beta-galactosidase antibodies as ligand, followed by an anion-exchange column. It was used for the purification and concentration of recombinant fusion proteins from Mycobacterium tuberculosis and M. leprae. Small amounts of crude lysates of Escherichia coli were loaded stepwise onto the immunoaffinity column with intermittent washing, elution and re-equilibration. After several cycles the eluate was passed through the anion-exchanger. Using an immunoaffinity gel of 5-ml volume and the anion-exchanger Mono Q HR 5/5, from 10 ml of crude E. coli lysate (containing up to 50 mg of protein) up to 100 micrograms of recombinant protein in a 2-ml volume could be isolated overnight.     

An Integrated Chemical Cytometry Method: Shining a Light on Akt Activity in Single Cells

Tools to evaluate oncogenic kinase activity in small clinical samples have the power to guide precision medicine in oncology. Existing platforms have demonstrated impressive insights into the activity of protein kinases, but these technologies are unsuitable for the study of kinase behavior in large numbers of primary human cells. To address these limitations, we developed an integrated analysis system that utilizes a light‐programmable, cell‐permeable reporter deliverable simultaneously to many cells. The reporter's ability to act as a substrate for Akt, a key oncogenic kinase, was masked by a 2‐4,5‐dimethoxy 2‐nitrobenzyl (DMNB) moiety. Upon exposure to ultraviolet light and release of the masking moiety, the substrate sequence enabled programmable reaction times within the cell cytoplasm. When coupled to automated single‐cell capillary electrophoresis, statistically significant numbers of primary human cells were readily evaluated for Akt activity.     

Pretreatment of Hardwood and <Emphasis Type="Italic">Miscanthus</Emphasis> with <Emphasis Type="Italic">Trametes versicolor</Emphasis> for Bioenergy Conversion and Densification Strategies

Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12–24 h. The amount of GCD in medium increased until 60–84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells.     

Maximizing automation in LC/MS high-throughput analysis and purification

Here, we describe a system for LC/MS-based analysis and purification of compounds aiming at the minimization of manual interference in the overall process. Key elements of the concept are automated identification of the target compounds, automated assignment of optimized preparative gradients for purification of the target compounds, and automated purity assessment of fractions with subsequent pooling of validated product fractions. Additional support is provided by an automated solvent and waste management system. One person can easily process 100-200 compounds on a 150-mg scale per day on that system, while still the maximization of purity and yield after purification is guaranteed. Reduced demands with respect to purity or yield can lead to significantly higher throughput numbers.     

Recombinant Production and Characterization of SAC,the Core Domain of Par-4, by SUMO Fusion System

Prostate apoptosis response-4 (Par-4), an anticancer protein that interacts with cell surface receptor GRP78, can selectively suppress proliferation and induce apoptosis of cancer cells. The core domain of Par-4 (aa 137–195), designated as SAC, is sufficient to inhibit tumor growth and metastasis without harming normal tissues and organs. Nevertheless, the anticancer effects of SAC have not been determined in ovarian cancer cells. Here, we developed a novel method for producing native SAC in Escherichia coli using a small ubiquitin-related modifier (SUMO) fusion system. This fusion system not only greatly improved the solubility of target protein but also enhanced the expression level of SUMO-SAC. After purified by Ni-NTA affinity chromatography, SUMO tag was cleaved from SUMO-SAC fusion protein using SUMO protease to obtain recombinant SAC. Furthermore, we simplified the purification process by combining the SUMO-SAC purification and SUMO tag cleavage into one step. Finally, the purity of recombinant SAC reached as high as 95% and the yield was 25 mg/L. Our results demonstrated that recombinant SAC strongly inhibited proliferation and induced apoptosis in ovarian cancer cells SKOV-3. Immunofluorescence analysis and competitive binding reaction showed that recombinant SAC could specifically induce apoptosis of SKOV-3 cells through combination with cell surface receptor, GRP78. Therefore, we have developed an effective strategy for expressing bioactive SAC in prokaryotic cells, which supports the application of SAC in ovarian cancer therapy.     

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.     

Self-cleavable stimulus responsive tags for protein purification without chromatography

A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.     

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome and peptide profiling analysis by using a monolithic analytical capillary column

An automated nano-LC-MS/MS platform without trap column was established, which only used a 20 cm lauryl methacrylate-ethylene dimethacrylate (LMA-EDMA) monolithic capillary column to allow preconcentration and separation of peptides. The monolithic column had the advantages of good permeability and low backpressure resulting in higher flow rates for capillary columns. Tryptic digests of bovine albumin and yeast protein extract were tested using the monolithic column system. High proteomic coverage using this approach were demonstrated in this study. Furthermore, peptide samples extracted from mouse liver were separated by using the monolithic column system combined with size-exclusion chromatography prefractionation. This monolithic column system might be a promising alternative for the automated system previously using a trap column for routine proteome and peptide profiling analysis.     

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.     

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system

Inteins are self-cleavable proteins that under reducing conditions can be cleaved from a recombinant target protein. Industrially, an intein-based system could potentially reduce production costs of recombinant proteins by facilitating a highly selective affinity purification using an inexpensive substrate such as chitin. In this study, SuperPro Designer was used to simulate the large-scale recovery of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification process based on the commercially available IMPACT system. The intein process was also compared with a conventional process simulated by SuperPro. The intein purification process initially simulated was significantly more expensive than the conventional process, primarily owing to the properties of the chitin resin and high reducing-agent (dithiothreitol [DTT]) raw material cost. The intein process was sensitive to the chitin resin binding capacity, cleavage efficiency of the intein fusion protein, the size of the target protein relative to the intein tag, and DTT costs. An optimized intein purification process considerably reduced costs by simulating an improved chitin resin and alternative reducing agents. Thus, to realize the full potential of intein purification processes, research is needed to improve the properties of chitin resin and to find alternative, inexpensive raw materials.     

Design of Recombinant Stem Cell Factor–macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity <Emphasis Type="BoldItalic">In Vitro</Emphasis>

Summary Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF–M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF–M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1–165aa) and M-CSF (1–149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of ~84 KD under non-reducing conditions and a monomer of ~42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.     

Electrophoretic immunodesorption of proteins according to molecular weight by use of a double-membrane system

Summary A simple method is described for electrophoretic desorption of proteins from antigen-antibody complexes, with more than 90% recovery and without denaturation, after immunosorbent affinity chromatography. Radiolabeled or unlabeled human serum albumin (HSA) and α-1-antitrypsin (AAT), conjugated to rabbit anti-HSA or anti-AAT polyclonal antisera, respectively, were electrophoretically desorbed from Sepharose 4B. In addition, purification and concentration of the major HSA protein band (monomer) of 68 kD from the other oligomeric protein bands were achieved by use of a two-membrane system in a simple electroelution apparatus. The system consisted of an upper cellulose acetate membrane, with pore size 20 nm and separation limit 70 kD, and a lower dialysis cellophane membrane with molecular weight cut-off from 1–50 kD that cnables separation according to size. Furthermore, purification of the monomer HSA or AAT from normal human serum was performed with 92% recovery. Homogeneity was implied by the presence of one band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Western blot, and autoradiography.