Bioseparation using affinity techniques.

Bioseparation of proteins from dilute solutions using different novel affinity procedures is reviewed. Emphasis is also placed on the quality of the product separated. Whenever possible, physical insights into the separation procedure are provided, besides indicating suitable directions where appropriate further research may be carried out. The procedures analyzed are different affinity chromatographic techniques, affinity separation using liquid perfluorocarbon supports, water soluble nonionic surfactants for affinity bioseparations, affinity cross-flow filtration, bioaffinity separation using reversed micelles, affinity precipitation and dual-functional affinity protein purification.     

蛋白质纯化技术研究进展

蛋白质组学研究的基础就是蛋白质的分离。对于天然蛋白来说,可能需要一系列的纯化步骤才能获得纯度满足研究要求的蛋白质,但是蛋白质在分离过程中常常由于溶液环境变化或外力作用造成构象变化而引起失活。本文首先介绍了常用的蛋白质分离纯化技术及其研究进展,包括膜分离技术、沉淀分离技术、电泳分离技术以及层析分离技术等常用的蛋白质纯化技术,总结了现有技术存在的问题,并对近年来发展的新型蛋白质分离技术--非对称流场流分离技术进行了介绍和展望。     

Free-solution isoelectric focusing for the purification of Staphylococcus aureus enterotoxin C1

A free-solution isoelectric focusing protocol was developed for the preparative purification of Staphylococcus aureus enterotoxin C1 (SEC1). A toxin consisting of a single isoelectric species, pI 8.8, was purified. Thirty-nine milligrams of SEC1 was recovered from 3 liters of culture supernatant. This significantly improved purification scheme utilized ammonium sulfate precipitation and the Bio-Rad Rotofor isoelectric cell to complete isolation in 2 days, thereby avoiding the protein degradation prevalent when published procedures are used. The purification protocol developed here for SEC1 is used to illustrate the utility of Rotofor fractionation in the general purification of bacterial exotoxins.     

Preparative parallel protein purification (P4)

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.     

Metal chelate affinity precipitation: a new approach to protein purification

Metal chelate affinity precipitation of proteins, a method combining metal–protein interaction and affinity precipitation is being discussed as a selective separation process for proteins. The technique utilizes a flexible soluble–insoluble thermo-responsive polymer with a covalently linked ligand loaded with metal ions. The affinity binding of the target protein varies with different metal ions. Copolymers of N-isopropylacrylamide with 1-vinylimidazole loaded with Cu(II) ions are designed as a potential carriers for affinity purification and proved to be successful for purification of protein inhibitors from a variety of cereals. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cellular retinoic acid-binding protein from rat testis. Purification and characterization

Cellular retinoic acid-binding protein has been purified to homogeneity from rat testes. The procedures utilized in the purification included acid precipitation, gel filtration, and chromatography on DEAE-cellulose. The binding protein was purified approximately 12,000-fold, based on total soluble testicular protein. The protein is a single polypeptide chain with a molecular weight of 14,600, determined by information from gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinoic acid with high affinity; the apparent dissociation constant was determined by fluorometric titration to be 4.2 X 10(-9) M.     

Purification of several proteins involved in glycogen metabolism

Several well-established procedures for the isolation of enzymes involved in glycogen metabolism have been modified such that all the enzymes can now be isolated from the same muscle preparation. The purified proteins are the catalytic subunit of cyclic AMP-dependent protein kinase, its thermostable inhibitor, glycogen phosphorylases a and b, and phosphorylase kinase. Phosphorylase kinase is separated by acid precipitation of the muscle extract. The other proteins are purified from the acid supernatant by chromatography on DEAE-cellulose. Further purification of each protein to homogeneity is then achieved using previously described methods. The proposed protocol saves sample tissue, and considerably reduces the work involved in obtaining muscle samples.     

Expression and purification of recombinant and native calreticulin.

Calreticulin is a 60-kDa Ca(2+)-binding protein of the endo(sarco)plasmic reticulum membranes of a variety of cellular systems. The protein binds approximately 25 mol of Ca2+ with low affinity and approximately 1 mol of Ca2+ with high affinity and is believed to be a site for Ca2+ binding/storage in the lumen of the endo(sarco)plasmic reticulum. In the present study, we describe purification procedures for the isolation of recombinant and native calreticulin. Recombinant calreticulin was expressed in Escherichia coli, using the glutathione S-transferase fusion protein system, and was purified to homogeneity on glutathione-Sepharose followed by Mono Q FPLC chromatography. A selective ammonium sulfate precipitation method was developed for the purification of native calreticulin. The protein was purified from ammonium sulfate precipitates by diethylaminoethyl-Sephadex and hydroxylapatite chromatography procedures, which eliminates the need to prepare membrane fractions. The purification procedures reported here for recombinant and native calreticulin yield homogeneous preparations of the proteins, as judged by the HPLC reverse-phase chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified native and recombinant calreticulin were identified by their NH2-terminal amino acid sequences, by their Ca2+ binding properties, and by their reactivity with anticalreticulin antibodies.     

Protein purification and crystallization artifacts: The tale usually not told

The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non‐reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.     

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.     

A new biochemical engineering approach to the fractional precipitation of proteins

A biochemical engineering framework for optimizing the design and operation of fractional protein precipitation has been developed. The method utilizes a fractionation diagram to represent the purification of a product protein relative to total contaminating protein. The purification factor for a single or double-cut fractional precipitation is obtained as the gradient of an appropriate operating tie-line. A computer algorithm has been devised to maximize the tie-line gradient for a given yield enabling a plot of optimum purification factor versus yield to be constructed. The recovery of the enzyme alcohol dehydrogenase from clarified bakers homogenate using saturated ammonium sulphate has been examined. Fractionation and purification versus yield diagrams were used to investigate the effects of such process parameters as pH, temperature, and initial total protein concentration on fractionation efficiency. The results are discussed in terms of the underlying solubility and mixing phenomena and the industrial application of fractional precipitation.     

Cationic polymer precipitation for enhanced impurity removal in downstream processing

Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation–depth filtration and the current platform process for the purification of biologics.     

Optimum conditions for production and purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae

Several prophylactic human papillomavirus (HPV) vaccines have been developed based on virus-like particles (VLPs) made from viral L1 proteins. A substantial number of VLPs is necessary for biochemical characterization and diagnostic test development. To establish the optimum conditions for production and purification of HPV L1 in the yeast expression system we varied the amount and nature of the carbon source and evaluated HPV 16 L1 recovery by three purification methods. Maximally threefold more HPV 16 L1 was produced with a 4% carbon source than with a 2% carbon source. In addition, the productivity of HPV 16 L1 varied by 25% depending on the combination of glucose and galactose in the 4% carbon source. We introduced an ammonium sulfate precipitation step in place of the ultracentrifugation using a sucrose cushion routinely used for HPV L1 purification, and optimized the purification by cation-exchange chromatography. Overall L1 protein recovery using the ammonium sulfate precipitation method was 30%, the highest recovery achieved so far. The purified HPV 16 L1 protein successfully self-assembled into VLPs. Purification by ammonium sulfate precipitation was maximally 15 times greater than ultracentrifugation on a sucrose cushion. We anticipate that our procedures for production and purification will reduce the cost, time and labor involved in obtaining sufficient yields of VLPs.     

Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography

Calmodulin binds quantitatively to phenyl-Sepharose and octyl-Sepharose affinity columns in the presence of micromolar concentrations of Ca²⁺. In addition to EGTA, calmodulin also can be eluted from these affinity columns with low ionic strength buffer, non-ionic detergent (i.e., 1% Triton X-100), or ethylene glycol (50%), suggesting hydrophobic interaction. Using hydrophobic interaction chromatography calmodulin can be purified to homogeneity from bovine brain homogenate in a single step. For large-scale purification the protein fraction containing calmodulin was concentrated by isoelectric precipitation prior to application to the affinity column. The yield obtained by this procedure (160–180 mg calmodulin per kg brain) is significantly greater, and the time required (～ 5 hr) is substantially less, than that of previously described procedures for calmodulin purification. It is apparent that phenyl-Sepharose offers several advantages over phenothiazine-Sepharose for affinity purification of calmodulin.     

Partial purification of Cytochrome P450 from Helicoverpa armigera (Hübner)

Abstract The cytochrome P450 (Cyt‐P450) proteins from the fat body and midgut of the cotton bollworm, Helicoverpa armigera, were respectively partially purified by a set of purification procedures including differential centrifugation, solubilization of CHAPS, protein precipitation by PEG precipitation and DE‐32 column chromatography. The Cyt‐P450 was detected by methods of CO difference spectrum and SDS‐PAGE. Fraction of detergent solubilized microsomes from the fat body of H. armigera was purified more than 17‐fold. Three protein bands were detected by SDS‐PAGE with molecular masses of 70 600, 63 300 and 571 200Da. It is possible that the proteins with molecular mass of 63 300 and 571 200Da were the isozymes of Cyt‐P450.     

Purification of recombinant protein by cold‐coacervation of fusion constructs incorporating resilin‐inspired polypeptides

Polypeptides containing between 4 and 32 repeats of a resilin‐inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence‐related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold‐coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein‐poor phase. We show that purification of recombinant proteins by cold‐coacervation can be performed when engineered as a fusion partner to a resilin‐inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time‐consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold‐coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. Biotechnol. Bioeng. 2012; 109: 2947–2954. © 2012 Wiley Periodicals, Inc.     

Proteins associated with tubulin.

The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H₁) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

Cellular retinol-binding protein from rat liver. Purification and characterization

Cellular retinol-binding protein has been purified to homogeneity from rat liver. The procedures utilized in the purification included acid precipitation, gel filtration on Sephadex G-75 and G-50, and chromatography on DEAE-cellulose. The binding protein was purified approximately 3,500-fold, based on total soluble liver protein. The protein is a single polypeptide chain with a molecular weight of 14,600 based on information obtained by the techniques of sedimentation equilibrium analysis, gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinol with high affinity; the appparent dissociation constant was determined by fluorometric titration to be 1.6 X 10(-8) M. Retinol bound to the protein has an absorption spectrum (lambdamax, 350 nm) considerably altered from the spectrum of retinol in ethanol (lambdamax, 325 nm).