Perfusion reversed-phase high-performance liquid chromatography for protein separation from detergent-containing solutions: an alternative to gel-based approaches

Detergents are frequently used for the solubilization of membrane proteins during and after purification steps. Unfortunately some of these detergents impair chromatographic separations and mass spectrometry (MS) analysis. Perfusion reversed-phase high-performance liquid chromatography (RP-HPLC) using POROS materials is suited for separating intact proteins solubilized by detergents due to the particles' highly diffusive pores and chemical stability. In this article, the use of perfusive reversed-phase material packed into small inner diameter capillary columns is presented as a cheap, rapid, and efficient method for the removal of different types of detergents from protein solutions. The ability to purify and separate the subunits of membrane protein complexes with self-packed capillary columns is exemplified for bovine cytochrome bc(1) complex. Even highly hydrophobic subunits can be detected in collected fractions by intact mass measurements and identified after proteolytic digestion and matrix-assisted laser desorption/ionization tandem MS (MALDI MS/MS). The comparison with a gel-based approach shows that this method is a valuable alternative for purification and separation of intact proteins with subsequent MS analysis and that hydrophobic proteins are even better represented in the LC-based approach.     

Use of ZetaPrep cartridge for the purification of human recombinant interleukin 1 beta

Zetaprep mass ion-exchange media represent a rapid and efficient chromatographic tool in the separation of proteins, in place of the conventional agarose or cellulose-based gels. We adopted this method, combined with classical steps, to purify to homogeneity human recombinant interleukin 1 beta (IL-1 beta) produced from E. coli and from S. cerevisiae. An anion exchanger QAE-ZetaPrep was used to achieve a rapid partial purification of both proteins. The IL-1 beta purification was completed by gel permeation chromatography on Sephadex G-50. When the protein was produced from yeast, an intermediate chromatographic step on a hydroxylapatite column was also necessary. The isolated proteins proved to be homogeneous by electrophoresis and amino acid analysis. The biological activity of IL-1 beta produced by E. coli is comparable to that of the natural protein, while the protein produced by yeast showed very low specific activity.     

Discrimination between stable and dynamic components of protein complexes by means of quantitative proteomics

To discriminate between stable and dynamic protein-protein interactions, we propose a strategy in which cells with and without tagged bait are differentially labeled with stable isotope and combined prior to complex purification. Mass-spectrometric analysis of the purified complexes identifies stable and dynamic components as those derived exclusively from the tagged cells and those from both cells, respectively. We successfully applied this strategy to analyze two yeast protein complexes, eIF2B-eIF2 and cyclin-Cdc28.     

Demonstration of robust host cell protein clearance in biopharmaceutical downstream processes

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.     

Use of a Label-Free Quantitative Platform Based on MS/MS Average TIC to Calculate Dynamics of Protein Complexes in Insulin Signaling

A label-free quantification strategy including the development of in-house software (NakedQuant) to calculate the average TIC across all spectral counts in tandem affinity purification (TAP)-tagging liquid chromatography-mass spectrometry MS/MS (LC/MS/MS) experiments was applied to a large-scale study of protein complexes in the MAPK portion of the insulin signaling pathway from Drosophila cells. Dynamics were calculated under basal and stimulating conditions as fold changes. These experiments were performed in the context of a core service model with the user performing the TAP immunoprecipitation and the MS core performing the MS and informatics stops. The MS strategy showed excellent coverage of known components in addition to potentially novel interactions.     

Is there a rational method to purify proteins? From expert systems to proteomics

The purification of recombinant proteins for therapeutic or analytical applications requires the use of several chromatographic steps in order to achieve a high level of purity. A range of techniques is available such as anion and cation exchange chromatography, which can be carried out at different pHs, and hence used at different steps, hydrophobic interaction chromatography, gel filtration and affinity chromatography. Evidently when confronted with a complex mixture of partially unknown proteins or a clarified cell extract there are many different routes one can take in order to choose the minimum and most efficient number of purification steps to achieve a desired level of purity (e.g. 98, 99.5 or 99.9%). In this review we will show how an initial "proteomic" characterization of the complex initial mixture of target protein and protein contaminants can be used to select the most efficient chromatographic separation steps in order to achieve a maximum level of purity with a minimum number of steps. The chosen methodology was implemented in a computer based expert system. The first algorithm developed was used to select the most efficient purification method to separate a protein from its contaminants based on the physicochemical properties of the protein product and the protein contaminants. The second algorithm developed was used to predict the number and concentration of contaminants after each separation as well as protein product purity. The successful application of the expert system approach, based on an initial proteomic characterization, to the practical cases of protein mixtures and clarified fermentation supernatant is presented and discussed. The purification strategy proposed was experimentally tested and validated with a mixture of four proteins and the experimental validation was also carried out with an "unknown" supernatant of Bacillus subtilis producing a recombinant beta-1,3-glucanase. The system was robust to errors <10% which is the range that can be found in the experimental determination of the properties in the database of product and contaminants. On the other hand, the system was sensitive both to larger variations (>20%) in the properties of the contaminant database and the protein product and to variations in one protein property (e.g. hydrophobicity).     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.     

Intein-mediated rapid purification of Cre recombinase

Cre recombinase produced by bacteriophage P1 catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps. In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein. The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column. Using the intein system, we have obtained highly pure nontagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification. The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months.     

Phosphoprotein profiling by PA-GeLC-MS/MS

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.     

Combination of MS Protein Identification and Bioassay of Chromatographic Fractions to Identify Biologically Active Substances from Complex Protein Sources

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C₄ reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.Purification and identification of biologically active proteins existing in minute amounts from biological sources such as urine is still a difficult task (1). It requires a large volume of the sample and many separation steps for purification (2, 3). Nevertheless the recent progress of MS has dramatically changed protein analysis (4). With MS, smaller protein samples can be used than with classical protein identification methods such as N-terminal peptide sequencing.Interstitial cystitis (IC)¹ is a chronic inflammatory disease characterized by frequency and urgency and/or severe pelvic pain (5). The International Continence Society also selected the term “painful bladder syndrome” for IC (6). The quality of life of IC patients is extremely low because of their severe symptoms. The pathogenesis of IC is unclear, and effective treatments have not been established. To elucidate the mechanism of IC pathogenesis, we attempted to find characteristic proteins in IC urine using proteomics techniques and have already reported active neutrophil elastase as an IC urinary marker (7). We had also performed gene expression analysis of IC bladder tissues using GeneChip technology and found that mRNA expression of GPR18, a member of the G-protein-coupled receptors, was higher in IC bladder than in the control.² We tried to confirm whether GPR18 endogenous ligand existed in IC urine by using a bioassay with GPR18 transfectant cells.In the present study, the existence of an active substance in IC urine was suggested in the bioassay using the serum response element (SRE)-dependent luciferase reporter gene with the stable recombinant HEK293 cell line expressing GPR18. We thought that the response was derived from GPR18 and tried to purify the active substance from a small volume of IC urine using chromatographic techniques. Among the many proteins identified from partially purified samples, we clearly nominated epidermal growth factor (EGF) as a candidate molecule judging from the correlation between MS protein identification and the bioassay of chromatographic fractions. With recombinant EGF and anti-EGF antibody, EGF was confirmed to be the desired substance found in IC urine. The complete inhibition of the bioassay response by anti-EGF receptor antibody also indicated that the response was based on the EGF receptor, not GPR18, suggesting that GPR18 overexpression enhanced the EGF signal via the endogenous EGF receptor of the HEK293 cell line.     

Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC

The proteome of any system is a dynamic entity, such that the intracellular concentration of a protein is dictated by the relative rates of synthesis and degradation. In this work, we have analyzed time-dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol we refer to as "dynamic SILAC", using 1-D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells, requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment. As turnover rates are acquired, a profile can be built up that allows exploration of the 'dynamic proteome' and of putative features that predispose a protein to a high or a low rate of turnover. Moreover, measurement of the turnover rate of individual components of supramolecular complexes provides a unique insight in processes of protein complex assembly and turnover.     

Horseradish esterases: detection,purification and identification

Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purification and identification of esterases from different horseradish tissues: plantlets and two tumor tissue lines. Horizontal IEF system enabled separation of six esterase isoforms with quite different pI values as well as with pronounced differences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel electrophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purification and characterization. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in-solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identification of two esterase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1.     

A Proteomic Strategy for Global Analysis of Plant Protein Complexes

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.     

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.     

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.     

Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli.

The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies.     

包涵体蛋白的分离和色谱法体外复性纯化研究进展

重组蛋白在大肠杆菌中表达多为无活性的包涵体形式,须经洗涤、溶解、复性后才能得到生物活性蛋白。综述了近年来包涵体蛋白分离纯化和复性技术研究进展,重点讨论了色谱法复性技术的应用,包括尺寸排阻色谱、亲和色谱、离子交换色谱、疏水相互作用色谱、固定化脂质体色谱、扩张床吸附色谱的进展情况。     

Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks

Most current methods for purification and identification of protein complexes use endogenous expression of affinity-tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, and gel separation and in-gel digestion prior to mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pull-down assay with denaturing elution, trypsin digestion in organic solvent, and LC-ESI MS/MS protein identification using SEQUEST analysis. Our method is simple and easy to scale-up and automate, making it suitable for high-throughput mapping of protein interaction networks and functional proteomics.