In-process monitoring of protein purification with thin film silicon sensor technology

We have developed a rapid and sensitive thin film assay for in-process monitoring of target protein purification. This novel biosensor method provides rapid (5-min) visual evaluation of column purification fractions. The method can be used to monitor the efficiency of purification and potential loss of protein if the column binding capacity is exceeded. The eluted fractions containing the highest yield of target protein can be quickly identified, pooled, and processed. This convenient platform, known as the SILAS product, is a thin-film detection technology in which specific molecular interactions are transduced into visible color changes based on changes in the optical thickness of layers on a silicon surface. The results are interpreted without instrumentation. Proteins eluted from a purification column are adsorbed to the assay surface, and the ligand of interest (target) can be identified with specific binding reagents. Here we demonstrate two protein purification applications for the SILAS technology product: monitoring antibody elution from a Protein G column and evaluating the efficiency of purification of a glutathione-S-transferase (GST)-tagged recombinant protein through each step of the purification process.     

The tandem affinity purification (TAP) method: a general procedure of protein complex purification

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.     

A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified protein. The advantage of this method is that thrombin is used instead of imidazole in the final purification step. Imidazole can influence NMR experiments, competition studies, or crystallographic trials, and the presence of imidazole often results in protein aggregates. Removal of the His-tag results in a form of the protein of interest in which no additional tags are present, resembling the native form of the protein, with only three additional amino acids at the N-terminal side. Our method is compared with a more conventional method for the purification of the Azotobacter vinelandii NIFL PAS domain, overexpressed in Escherichia coli. It also proves to be successful for three different His-tagged proteins, the Klebsiella pneumoniae NTRC protein, and the A. vinelandii NIFA and NIFL proteins, and therefore it is a general method for the purification of His-tagged proteins.     

Tandem affinity purification of functional TAP-tagged proteins from human cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.     

Simple procedures for analyzing and purifying methylene green

Methylene green is a versatile dye that can be used in a wide range of technical applications, most of which require the dye to be pure. Because commercial lots of methylene green are known to be heterogeneous, we report a thin layer chromatographic method for checking purity. We also describe a simple and effective flash chromatographic purification procedure for subsequent purification. The identity and purity of the dye can be checked easily using UV-visible absorption spectrum measurements or by more sophisticated procedures if necessary.     

Purification of supercoiled plasmids from crude cell lysates using high performance anion exchange chromatography

High performance liquid chromatography is of increasing importance in the purification of nucleic acids. Recently, a new anion exchange column called Gen-Pak FAX has been introduced for this purpose. Previously, it has been used in the purification of restriction fragments and oligonucleotides. In this paper we present the use of the Gen-Pak FAX column for the purification of plasmids from crude E. coli lysates. The different conformational forms of the plasmid can be well separated and collected with high recoveries of both mass and activity. Up to 50 micrograms of supercoiled plasmid can be purified in a single 30 min run with up to 98% purity.     

Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350–sodium citrate aqueous two-phase systems (ATPS) was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, sodium citrate and NaCl concentrations of 15.1, 11.0 and 8.52% respectively. However, under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.     

Purification of Proteins by the Use of Hydrophobic Zeolite Y

Hydrophobic zeolite Y can be used as a fast and efficient and inexpensive matrix in the purification of proteins from crude extracts. Preferably the zeolite can be used in the first purification step, replacing the commonly used precipitation techniques with (NH₄)₂SO₄ or ethanol. The time required for the zeolite prefractionation was a few hours compared to the much more time consuming precipitation procedure which demands centrifugation and subsequent dialysis. Proteins can be adsorbed on the zeolite either in order to remove undesired proteins or to be subsequently eluted from the zeolite in order to achieve purification and concentration. Removal of undesired proteins is exemplified by the purification of horseradish peroxidase from a crude extract. The zeolite procedure enhanced the specific activity five times and provided a yield similar to that which was obtained by the use of standard procedures, (NH₄)₂SO₄ fractionation and ion-exchange chromatography. Binding and subsequent elution of proteins from the zeolite is exemplified by the purification of monoclonal antibodies from hybridoma culture supernatants. Proteins were desorbed from the zeolite by the use of polyethylene glycol 600 and this procedure yielded a purification factor of 5.     

Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes

Peptides can be labeled with various trivalent radiometals for imaging or targeted radionuclide-therapy applications. The peptide is first conjugated to a chelating agent that is able to form stable complexes with the radionuclide of interest. This conjugation step can be carried out as part of the solid-phase peptide synthesis, or it can be undertaken in the solution phase after synthesis and purification of the peptide. The latter route, described here, involves reacting a molar excess of the activated tri-tert-butyl ester-derivatized chelator with a designated free amino group of a peptide analog, in which all other reactive amines are protected, in the presence of a coupling agent. The conjugate molecule is then purified prior to deprotection and further purification by HPLC. The product can be radiolabeled by addition of a suitable metal salt, followed, if necessary, by removal of the unchelated metal. The entire process of conjugation, purification and radiolabeling should take approximately 12.5 h.     

Simple procedures for analyzing and purifying methylene green

Abstract

Methylene green is a versatile dye that can be used in a wide range of technical applications, most of which require the dye to be pure. Because commercial lots of methylene green are known to be heterogeneous, we report a thin layer chromatographic method for checking purity. We also describe a simple and effective flash chromatographic purification procedure for subsequent purification. The identity and purity of the dye can be checked easily using UV-visible absorption spectrum measurements or by more sophisticated procedures if necessary.     

Large-scale purification of oligonucleotides by extraction and precipitation with butanole

Today the synthesis of oligonucleotides is a well-established process. Using automatic synthesizers even kilogram quantities can be produced in a few hours. However, the purification of the final product is still time-consuming and needs a complex apparatus. In this article, a simple and fast purification method for the large-scale syntheses of oligonucleotides is described. According to the method of Sawadago and van Dyke ([1991] Nucleic Acids Res 19:674-675) for small-scale oligonucleotide purification, oligonucleotides in mumol to mmol amounts were purified by liquid-liquid extraction using butanole as the extraction liquid. Choosing appropriate ratios of extraction liquid to oligonucleotide solution, simultaneous purification and precipitation could be achieved. It was found that the yield of the purified oligonucleotide was mainly affected by the temperature. Yield decreased with increasing temperature. The use of this improved extraction procedure allows the purification of gram to kilogram quantities of oligonucleotides in less than a day with simple equipment and high yield.     

Integrated recombinant protein expression and purification platform based on Ralstonia eutropha

Protein purification of recombinant proteins constitutes a significant cost of biomanufacturing and various efforts have been directed at developing more efficient purification methods. We describe a protein purification scheme wherein Ralstonia eutropha is used to produce its own "affinity matrix," thereby eliminating the need for external chromatographic purification steps. This approach is based on the specific interaction of phasin proteins with granules of the intracellular polymer polyhydroxybutyrate (PHB). By creating in-frame fusions of phasins and green fluorescent protein (GFP) as a model protein, we demonstrated that GFP can be efficiently sequestered to the surface of PHB granules. In a second step, we generated a phasin-intein-GFP fusion, wherein the self-cleaving intein can be activated by the addition of thiols. This construct allowed for the controlled binding and release of essentially pure GFP in a single separation step. Finally, pure, active beta-galactosidase was obtained in a single step using the above described method.     

Rapid purification of annexin V from human placenta by affinity chromatography

Affinity purification of annexin V from human placenta on column with appropriate monospecific antibodies is developed. The procedure permits purification of the protein to a highly purified state by a two stage procedure. The yield of the protein is about 5 mg per 100 g of wet tissue. Because of high homologies between various annexins, it was supposed that this procedure can be also applied for purification of other annexins from other tissues.     

Purification of rat hepatic glucokinase

We have developed a rapid, reliable procedure for the purification of rat hepatic glucokinase. The purification utilizes DEAE-cellulose, two affinity chromatography steps, and high-performance liquid chromatography. Glucokinase with a specific activity of 240 units/mg, a 42 K-fold purification, and a yield of 60% is obtained. The enzyme appears as a homogeneous band, with over 99% purity as assessed by polyacrylamide gel electrophoresis. The purification procedure can be completed in 5 days.     

Automated, High Productivity Purification and Analysis of Synthetic Peptides

Methods for peptide assembly consist of techniques that allow for construction of complex sequences. The advantage of solid-phase methodologies is automation of the repetitive processes of deprotecting, washing, and coupling protected amino acids (acylation). However, for difficult sequences the crude product contains a variety of side products that must be removed to provide the desired target peptide in sufficient concentration and purity. This paper illustrates that high efficiency purification method-development can be achieved by combining purification and analysis on a single platform. Incorporation of fast LC-based assays using polystyrene-based POROS® Perfusion Chromatography media permitted rapid overall processing times from crude peptide purification through fraction pooling and product verification. Application of these technologies to the purification of peptides at scales of 100 mg is demonstrated.     

Automated,High Productivity Purification and Analysis of Synthetic Peptides

Methods for peptide assembly consist of techniques that allow for construction of complex sequences. The advantage of solid-phase methodologies is automation of the repetitive processes of deprotecting, washing, and coupling protected amino acids (acylation). However, for difficult sequences the crude product contains a variety of side products that must be removed to provide the desired target peptide in sufficient concentration and purity. This paper illustrates that high efficiency purification method-development can be achieved by combining purification and analysis on a single platform. Incorporation of fast LC-based assays using polystyrene-based POROS® Perfusion Chromatography media permitted rapid overall processing times from crude peptide purification through fraction pooling and product verification. Application of these technologies to the purification of peptides at scales of 100 mg is demonstrated.     

Protein Purification with C-Terminal Fusion of Maltose Binding Protein

For affinity-chromatography-based purification of proteins that are prone to abnormal termination of translation or that may not be modified at their N-termini, affinity tags are needed which can be fused to the C-terminus. In this publication we describe that maltose binding protein (MBP) fused to the C-terminus of the plant photoreceptor phytochrome B allows purification of the fusion protein via amylose affinity chromatography. After overexpression in yeast a 125-fold enrichment could be achieved. The spectral properties of phytochrome B were not impaired by the fusion and purification. These results demonstrate that not only the widely used N-terminal fusions of MBP but also C-terminal fusions can be employed for protein purification.     

Multi-angle light scattering as a process analytical technology measuring real-time molecular weight for downstream process control

For many protein therapeutics including monoclonal antibodies, aggregate removal process can be complex and challenging. We evaluated two different process analytical technology (PAT) applications that couple a purification unit performing preparative hydrophobic interaction chromatography (HIC) to a multi-angle light scattering (MALS) system. Using first principle measurements, the MALS detector calculates weight-average molar mass, M_w and can control aggregate levels in purification. The first application uses an in-line MALS to send start/stop fractionation trigger signals directly to the purification unit when preset M_w criteria are met or unmet. This occurs in real-time and eliminates the need for analysis after purification. The second application uses on-line ultra-high performance size-exclusion liquid chromatography to sample from the purification stream, separating the mAb species and confirming their M_w using a µMALS detector. The percent dimer (1.5%) determined by the on-line method is in agreement with the data from the in-line application (M_w increase of approximately 2750 Da). The novel HIC-MALS systems demonstrated here can be used as a powerful tool for real-time aggregate monitoring and control during biologics purification enabling future real time release of biotherapeutics.     

Rapid preparation of the mitotic kinesin Eg5 inhibitor monastrol using controlled microwave-assisted synthesis

We present here a protocol for the synthesis of the dihydropyrimidine (DHPM) derivative monastrol, which is known to be a specific mitotic kinesin Eg5 inhibitor. By applying controlled microwave heating under sealed-vessel conditions, the synthesis via the one-pot three-component Biginelli condensation can be performed in a shorter reaction time (30 min) compared with conventional heating methods that normally require several hours of reflux heating. For the purification of the crude target compound, two different methods are presented. The first protocol includes a simple precipitation/filtration step to provide monastrol in 76% isolated yield and high purity so that no recrystallization step is necessary. This can be ascribed to the microwave heating technology in which less side-product formation is typically one of the advantages. In an alternative purification step, column chromatography is performed, which provides the product in a slightly higher yield (86%). Monastrol synthesis can be conducted in approximately 2 h by employing the precipitation/filtration purification method.     

Design, production, and characterization of an engineered biotin ligase (BirA) and its application for affinity purification of staphylokinase produced from Bacillus subtilis via secretion

A major attraction in using Bacillus subtilis as an expression host for heterologous protein production is its ability to secrete extracellular proteins into the culture medium. To take full advantage of this system, an efficient method for recovering the target protein is crucial. For secretory proteins which cannot be purified by a simple scheme, in vitro biotinylation using biotin ligase (BirA) offers an effective alternative for their purification. The availability of large amounts of quality BirA can be critical for in vitro biotinylation. We report here the engineering and production of an Escherichia coli BirA and its application in the purification of staphylokinase, a fibrin-specific plasminogen activator, from the culture supernatant of Bacillus subtilis via in vitro biotinylation. BirA was tagged with both a chitin-binding domain and a hexahistidine tail to facilitate both its purification and its removal from the biotinylated sample. We show in this paper how, in a unique way, we solved the problem of protein aggregation in the E. coli BirA production system to achieve a yield of soluble functional BirA hitherto unreported in the literature. Application of this novel BirA to protein purification via in vitro biotinylation in general will also be discussed. Biotinylated staphylokinase produced in the study not only can act as an intermediate for easy purification, it can also serve as an important element in the creation of a blood clot targeting and dissolving agent.