An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.     

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.     

A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.     

MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation

Enterovirus A71 (EV‐A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV‐A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV‐A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV‐A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV‐A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV‐A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID₅₀) values calculated in viral proliferations (ranged from 10^7.6 to 10^7.8); the TCID₅₀ being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV‐A71Jinan1002 amplifies more efficiently in MA104, Hep‐2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV‐A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV‐A71.     

Antibody variable region interactions with Protein A: implications for the development of generic purification processes

In this paper, a wide range of antibodies from various subclasses and subfamilies are employed to evaluate the creation of generic separation processes using Protein A chromatography. The reasons for elution pH differences amongst several IgG1s, IgG2s, antibody fragments, and Fc-fusion proteins during Protein A chromatography are investigated using several complimentary techniques. The results indicate that variable region interactions play a major role in determining elution pH for VH3 subfamily antibodies while using traditional protein A chromatographic materials. On the other hand, experiments with a resin which employs a ligand consisting solely of B domain of Protein A indicate that variable region interactions can be mitigated, enabling the use of a single elution pH for a range of antibodies. Finally, the moderation of elution conditions associated with this engineered ligand are shown to minimize problems associated with low pH induced aggregation. It is expected that the findings reported in this paper will facilitate faster process development cycle times for this important class of human therapeutics.     

Increase of protein synthesis after isolation in dormant and afterripened Agrostemma githago L. embryos

Isolated embryos are more suitable for in vivo study of protein synthesis than non-isolated embryos because, after isolation, the uptake of labeled amino acids is about 1000 times higher. However, isolation also stimulates protein synthesis: Up to 4 h after isolation, the capacity to incorporate labeled amino acids increased 7 times. Therefore, data on incorporation obtained with isolated embryos cannot be extended to non-isolated embryos. The increase of protein synthesis was not due to synthesis of specific proteins, but was a general increase. Furthermore, ripening, dormant, and afterripened embryos showed the same degree of increase. Isolation therefore stimulates protein synthesis nonspecifically. When embryos were kept under anaerobic conditions after isolation, protein synthesis did not increase. Therefore, higher oxygen consumption after removal of the seedcoat is probably the cause of the higher incorporation capacity. Furthermore, the activation of protein synthesis lagged several hours behind the increase of oxygen consumption.Abbreviations A afterripened - D dormant - pre-rRNA precursor of ribosomal RNA     

BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.     

A Mechanism of Global Shape-dependent Recognition and Phosphorylation of Filamin by Protein Kinase A

Protein phosphorylation mediates essentially all aspects of cellular life. In humans, this is achieved by ∼500 kinases, each recognizing a specific consensus motif (CM) in the substrates. The majority of CMs are surface-exposed and are thought to be accessible to kinases for phosphorylation. Here we investigated the archetypical protein kinase A (PKA)-mediated phosphorylation of filamin, a major cytoskeletal protein that can adopt an autoinhibited conformation. Surprisingly, autoinhibited filamin is refractory to phosphorylation by PKA on a known Ser²¹⁵² site despite its CM being exposed and the corresponding isolated peptide being readily phosphorylated. Structural analysis revealed that although the CM fits into the PKA active site its surrounding regions sterically clash with the kinase. However, upon ligand binding, filamin undergoes a conformational adjustment, allowing rapid phosphorylation on Ser²¹⁵². These data uncover a novel ligand-induced conformational switch to trigger filamin phosphorylation. They further suggest a substrate shape-dependent filtering mechanism that channels specific exposed CM/kinase recognition in diverse signaling responses.     

An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography

Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.     

Protein Kinase A Rescues Microtubule Affinity-regulating Kinase 2-induced Microtubule Instability and Neurite Disruption by Phosphorylating Serine 409

Microtubule affinity-regulating kinase 2 (MARK2)/PAR-1b and protein kinase A (PKA) are both involved in the regulation of microtubule stability and neurite outgrowth, but whether a direct cross-talk exists between them remains unclear. Here, we found the disruption of microtubule and neurite outgrowth induced by MARK2 overexpression was blocked by active PKA. The interaction between PKA and MARK2 was confirmed by coimmunoprecipitation and immunocytochemistry both in vitro and in vivo. PKA was found to inhibit MARK2 kinase activity by phosphorylating a novel site, serine 409. PKA could not reverse the microtubule disruption effect induced by a serine 409 to alanine (Ala) mutant of MARK2 (MARK2 S409A). In contrast, mutation of MARK2 serine 409 to glutamic acid (Glu) (MARK2 S409E) did not affect microtubule stability and neurite outgrowth. We propose that PKA functions as an upstream inhibitor of MARK2 in regulating microtubule stability and neurite outgrowth by directly interacting and phosphorylating MARK2.     

A method for isolation and purification of peanut genomic DNA suitable for analytical applications

Numerous methods are available for isolation of plant genomic DNA, but in practice these procedures are empirical due to variability in plant tissue composition. Consistent isolation of quality DNA from peanut (Arachis hypogaea L.) is particularly problematic due to the presence of phenolic compounds and polysaccharides. Inconsistencies in extraction results can be attributed to the age and growth stage of the plant material analyzed. Mature leaves have higher quantities of polyphenols, tannins, and polysaccharides that can contaminate DNA during isolation. We show that four published protocols could not be used to isolate peanut DNA of sufficient quality for PCR amplification or Southern hybridization. We have devised a new protocol that uses DEAE-cellulose purification to isolate peanut DNA useful for downstream applications.     

A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds

Myrosinase is a β-thioglucosidase glucohydrolase that catalyses the hydrolysis of the thioglucoside bond in glucosinolates, allelochemicals present in Brassicaceous plants. These isoenzymes have been found to form complexes with other proteins; however, traditional isolation procedures involving ammonium sulphate precipitation and/or ion exchange chromatography do not allow for the isolation of these complexes. The present paper reports a fast and gentle procedure for the isolation of myrosinases in the complex form. Partial purification by Con A affinity chromatography followed by Sephadex G-200 gel filtration allowed for the isolation of myrosinase complexes from seeds of Brassica carinata, B. oleracea var. capitata, B. napus and Sinapis alba. Myrosinases in the Brassicas formed complexes of different molecular weight (500–600 kDa, 270–350 kDa and 140–200 kDa) whereas in seeds of S. alba it was only possible to isolate and detect 140–200 kDa complexes. In all species the complexes were formed by isoenzymes with isoelectric points between 4.8 and 5.6 and in some cases up to 6.8. SDS-PAGE confirmed that the myrosinase isoenzymes were composed by several protein subunits of molecular weights ranging between 10 and 110 kDa. The relative amount and enzymatic activity of the myrosinase complexes varied amongst the species studied. The isolation of myrosinase complexes in their native form is of great importance for the study of the hydrolysis of glucosinolates under autolysis conditions.     

Anchored protein kinase A recruitment of active Rac GTPase

Protein kinase A-anchoring proteins (AKAPs) influence fundamental cellular processes by directing the cAMP-dependent protein kinase (PKA) toward its intended substrates. In this report we describe the identification and characterization of a ternary complex of AKAP220, the PKA holoenzyme, and the IQ domain GTPase-activating protein 2 isoform (IQGAP2) that is enriched at cortical regions of the cell. Formation of an IQGAP2-AKAP220 core complex initiates a subsequent phase of protein recruitment that includes the small GTPase Rac. Biochemical and molecular biology approaches reveal that PKA phosphorylation of Thr-716 on IQGAP2 enhances association with the active form of the Rac GTPase. Cell-based experiments indicate that overexpression of an IQGAP2 phosphomimetic mutant (IQGAP2 T716D) enhances the formation of actin-rich membrane ruffles at the periphery of HEK 293 cells. In contrast, expression of a nonphosphorylatable IQGAP2 T716A mutant or gene silencing of AKAP220 suppresses formation of membrane ruffles. These findings imply that IQGAP2 and AKAP220 act synergistically to sustain PKA-mediated recruitment of effectors such as Rac GTPases that impact the actin cytoskeleton.     

Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.     

Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.     

Protein disulphide isomerase-assisted functionalization of proteinaceous substrates

Protein disulphide isomerase (PDI) is an enzyme that catalyzes thiol-disulphide exchange reactions among a broad spectrum of substrates, including proteins and low-molecular thiols and disulphides. As the first protein-folding catalyst reported, the study of PDI has mainly involved the correct folding of several cysteine-containing proteins. Its application on the functionalization of protein-based materials has not been extensively reported. Herein, we review the applications of PDI on the modification of proteinaceous substrates and discuss its future potential. The mechanism involved in PDI functionalization of fibrous protein substrates is discussed in detail. These approaches allow innovative applications in textile dyeing and finishing, medical textiles, controlled drug delivery systems and hair or skin care products.     

适用于盐生植物的双向电泳样品制备方法

比较了三氯乙酸,丙酮沉淀法（TCA）、三氯乙酸沉淀法（E-TCA）和酚抽法（Phe）3种方法对盐生植物盐角草（Salicornia europaea L．）总蛋白的提取效果。3种方法分别得到579、343和535个蛋白点;TCA和E-TCA法所得图谱均存在严重的横向纹理,Phe法所得图谱则背景干净,基本上没有纹理。说明Phe法不仅能很好地提取盐角草蛋白,而且能有效去除样品中的盐分。对Phe法的提取液进行了改进,所得图谱背景更加清晰,蛋白点数增加。为其他盐生植物以及嗜盐微生物蛋白质的提取提供了重要参考。     

A Receptor-interacting Protein 1 (RIP1)-independent Necrotic Death under the Control of Protein Phosphatase PP2A That Involves the Reorganization of Actin Cytoskeleton and the Action of Cofilin-1

Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 and Drp1. However, G5, a non-selective isopeptidase inhibitor, triggers a distinct necrotic pathway that depends on the protein phosphatase PP2A and the actin cytoskeleton. PP2A catalytic subunit is stabilized by G5 treatment, and its activity is increased. Furthermore, PP2Ac accumulates into the cytoplasm during necrosis similarly to HMGB1. We have also defined in the actin-binding protein cofilin-1 a link between PP2A, actin cytoskeleton, and necrotic death. Cofilin-1-severing/depolymerization activity is negatively regulated by phosphorylation of serine 3. PP2A contributes to the dephosphorylation of serine 3 elicited by G5. Finally, a cofilin mutant that mimics phosphorylated Ser-3 can partially rescue necrosis in response to G5.