Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage

Purification tags are robust tools that can be used to purify a variety of target proteins. However, tag removal remains an expensive and significant issue that must be resolved. Based on the affinity and the trans-splicing activity between the two domains of Ssp DnaB split-intein, a novel approach for tag affinity purification of recombinant proteins with controllable tag removal by inducible auto-cleavage has been developed. This system provides a new affinity method and avoids premature splicing of the intein fused proteins expressed in host cells. The affinity matrix can be reused. In addition, this method is compatible with his-tag affinity purification technique. Our methods provide the insights for establishing a novel recombinant protein preparation system.     

Purification of recombinant proteins by chemical removal of the affinity tag

The efficient removal of a N-or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and aPlasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag inEscherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.     

Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose

A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined.     

A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles

A novel protein purification method was developed using microbial polyhydroxyalkanoates (PHA) granule-associated protein phasin, a pH-inducible self-cleaving intein and PHA nanoparticles. Genes for the target proteins to be produced and purified were fused to genes of intein and phasin, the genes were jointly over-expressed in vivo, such as in E. coli cells in this study. The fused proteins containing target protein, intein and phasin produced by the recombinant E. coli were released together with all other E. coli proteins via a bacterial lysis process. They were then adsorbed in vitro to the surfaces of the hydrophobic polymer nanoparticles incubated with the cell lysates. The nanoparticles attached with the fused proteins were concentrated via centrifugation. Then, the reasonably purified target protein was released by self-cleavage of intein and separated with nanoparticles by a simple centrifugation process. Using this system, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and beta-galactosidase were successfully purified in their active forms with reasonable yields, respectively, demonstrating the effectiveness and reliability of this purification system. This method allows the production and purification of high value added proteins in a continuous way with low cost.     

Novel affinity chromatography method for the efficient purification of recombinant Binder of SPerm homolog proteins

In mammalian species, a family of proteins named the Binder of SPerm proteins, which are expressed in the male reproductive tract, have been shown to play a role in epididymal sperm maturation and sperm capacitation. Recently, one homolog from human and two homologs from mouse were characterized. In order to further investigate the biochemical activity of these proteins, efficient purification procedures are required to isolate the proteins. Since these proteins are produced in very minute quantities, we exploited the high capacity of Escherichia coli to produce larger quantities of recombinant proteins that were subsequently purified using affinity chromatography on a diethylaminoethyl‐Sephadex A‐25 column. Binder of SPerm proteins have been shown to interact with pseudo‐choline groups such as diethylaminoethyl through affinity rather than ionic interactions. The aim of the current study was to develop a novel method for purifying these recombinant proteins, produced in Escherichia coli cells. Diethylaminoethyl is positively charged and is a weak anion exchanger, but binder of sperm proteins interacts with affinity to this resin. This study presents a new, rapid, and cost‐effective purification method that provides with an exceptional purity level, which can be used to study their roles in mammalian fertilization.     

Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography

Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50–60% range for affinity resins, and in the 80–85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.     

Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform

Single-step fusion-based affinity purification of proteins with pH-controllable linkers was carried out in a fluidic device. The linkers were previously derived from self-splicing protein elements called inteins. Two different linkers were generated to solve two distinct separation problems: one for rapid single-step affinity purification of a wide range of proteins, and the other specifically for the purification of cytotoxic proteins. Scale-down factors of 185 resulted in separations in a 27 microl bed-volume. A rotating CD format was chosen because of its simplicity in effecting fluid movement through centrifugal force without the complications associated with electro-osmosis and other pumping methods. The design and fabrication of the fluidic device and the protein purification process are described. This work, which demonstrates the purification of active proteins by two distinct fluidic separations, is widely applicable to small-scale massively parallel proteomic separations.     

Rational design of purification processes for recombinant proteins.

This paper discusses the elements important for rational design of purification processes for recombinant proteins. Main issues involved in selection of operations and process design are reviewed with particular emphasis on the challenges posed by recombinant proteins. This includes thermodynamic characterization of target protein and main contaminants, use of correlations and of expert knowledge for the development of an expert system for optimization and design (selection) of separation and purification (chromatographic) processes. The main deficiency in accurate information for rational process selection is in that required for high-resolution chromatographic processes. The authors show that a database with detailed information on properties of the main contaminants present in the fermentation streams of usual recombinant protein sources can be integrated to an expert system with an open architecture. This will allow more precise selection of unit operations for the design of protein purification processes.     

Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag peptides

Isotope-coded affinity tag (ICAT) methods, in conjunction with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS), represent a promising approach for accurate protein quantification. However, sensitivity remains a challenge for the quantification of low-copy proteins in complex biological matrices. Here we investigated the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides derivatized with the cleavable ICAT (cICAT) reagent. For cICAT-peptides that were either synthesized or obtained by digestion of model proteins, the cICAT moiety showed a tendency toward protonation under positive ESI, producing relatively intense triply charged cICAT-peptide ions ([IP+3H]3+). [IP+3H]3+ exhibited significantly higher CAD reactivity than did the doubly charged cICAT-peptide ([IP+2H]2+), and produced a greater abundance of fragments at lower collision energies. Fragmentation spectra of [IP+3H]3+ showed variable intensities of doubly charged y and b ions, and the amount of sequence information obtained was dependent on the position of the cICAT-labeled cysteine residue in the peptide sequence. However, the absolute abundances of major fragments of [IP+3H]3+ were much higher than for [IP+2H]2+. Although the efficiency of identification of cICAT-peptides was compromised by their charge distribution toward the triply charged state and by the unique CAD behavior of the [IP+3H]3+ ions, it was found that the triply charged ions provided higher sensitivity than [IP+2H]2+ for quantification using multiple reaction monitoring (MRM). ESI and CAD conditions for MRM of [IP+3H]3+ were optimized, and, for all cICAT-peptides studied, MRM using [IP+3H]3+ as precursors showed 2- to 8-fold higher sensitivity than obtained using [IP+2H]2+, without compromising quantitative accuracy. Using this approach, the time course of tyrosine aminotransferase induction by methylprednisolone was monitored in rat livers. A remarkably better signal-to-noise ratio was observed by using [IP+3H]3+ for quantification compared to [IP+2H]2+.     

Affinity purification and affinity characterization of carbohydrate-binding proteins in bovine kidney.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney by two-step affinity chromatography on fetuin and heparin columns and subsequent anion-exchange high-performance liquid chromatography. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the purified fraction gave two protein bands corresponding to proteins of relative molecular mass 33,000 (p33) and 41,000 (p41), respectively. Although the proteins had no haemogglutinating activities towards human and rabbit erythrocytes, their carbohydrate-binding activity was examined by a newly developed method using horseradish peroxidase (HRP) and/or biotin-labelled glycoconjugates as affinity probes. They could bind in a Ca(2+)-dependent manner to labelled fetuin and heparin in a specific and dose-dependent manner by solid-phase assay after immobilization on plastic plate surface. Inhibition assay of the binding revealed that N-acetylneuraminic acid is the most potent inhibitor of the proteins among the monosaccharides tested. Fucoidin and heparan sulphate most strongly inhibited the binding of the proteins to labelled heparin. Direct binding assay to acidic glycolipids prepared from bovine kidney showed that the proteins react with the ganglioside fraction but not with sulphatide [Gal(3-SO4) beta 1-1Cer]. These results indicated that the purified proteins have a significant affinity to charged oligosaccharides linking to glycoproteins, glycolipids and charged polysaccharides in a Ca(2+)-dependent manner.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Controlling the orientation of immobilized proteins on an affinity membrane through chelation of a histidine tag to a chitosan-Ni surface

The ability of histidine-tagged proteins to chelate to Ni⁺⁺ ions that are coordinated to functionalized polymeric matrices is the basis of affinity column separations. In this study, affinity membranes based on a Ni⁺⁺-chelating chitosan surface were fabricated to immobilize C-terminus hexahistidine-tagged green fluorescent protein (his-GFP). The binding of GFP antibody (antiGFP) to the immobilized his-GFP was measured and compared to a membrane with a chitosan surface to which his-GFP was immobilized through amine-glutaraldehyde chemistry as a control. Both membranes had comparable amounts of his-GFP immobilized on the surface. However, the amount of antiGFP bound to the Ni⁺⁺-chelated his-GFP at saturation was higher than that bound to the glutaraldehyde-immobilized his-GFP by a factor of five. Furthermore, fitting the data to a single-site Langmuir model resulted in an affinity constant for the Ni⁺⁺-chelated his-GFP towards antiGFP that was 14 times higher than the glutaraldehyde-immobilized his-GFP. The higher affinity suggests that immobilizing a protein at its C-terminus results in the proper orientation for subsequent antibody binding. At low antibody concentrations, the sensitivity of the affinity membrane is 70 times that of the control.     

Scale-up of recombinant protein purification by hydrophobic interaction chromatography

The scale-up of hydrophobic interaction chromatography is described. Human recombinant superoxide dismutase was used as a model. The scale-up was performed by keeping the height to diameter (H/D) ratio of the column constant. The success of scale-up was evaluated by reversed-phase high-performance liquid chromatography of the eluted material. The wrong H/D ratio causes decreased resolution.     

Metal ion affinity purification of proteins by genetically incorporating metal-chelating amino acids

Affinity tags are efficient tools for protein purification. They allow simple one-step purification of proteins to high purity. However, in some cases the tags cause structural and functional changes in a protein, and need to be removed. Therefore, affinity tags that are readily introduced into proteins with minimal perturbation and have specific affinity for purification are desired. Herein, two metal-chelating amino acids derived from 2,2′-bipyridine and 8-hydroxyquinoline were genetically incorporated into glutathione S-transferase (GST) and the mutant proteins were purified by using the metal ion affinity of the unnatural amino acids. The purification of the GST mutants containing 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) showed that the proteins could be efficiently enriched in Ni–NTA by the metal ion affinity of the unnatural amino acid and purified to excellent purity. This method should be very useful for general protein affinity purification, especially for proteins whose structure or function is affected by affinity tags fused to N- or C-terminals.     

A novel two-zone protein uptake model for affinity chromatography and its application to the description of elution band profiles of proteins fused to a family 9 cellulose binding module affinity tag

A novel two-zone model (TZM) is presented to describe the rate of solute uptake by the stationary phase of a sorption-type chromatography column. The TZM divides the porous stationary-phase particle into an inner protein-free core and an outer protein-containing zone where intraparticle transport is limited by pore diffusion and binding follows Langmuir theory. The TZM and the classic pore-diffusion model (PDM) of chromatography are applied to the prediction of stationary-phase uptake and elution bands within a cellulose-based affinity chromatography column designed to selectively purify proteins genetically labelled with a CBM9 (family 9 cellulose binding module) affinity tag. Under both linear and nonlinear loading conditions, the TZM closely matches rates of protein uptake within the stationary phase particles as measured by confocal laser scanning microscopy, while the PDM deviates from experiment in the linear-binding region. As a result, the TZM is shown to provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Synthesis of affinity chromatography and electrophoresis matrices for the purification of bile acid transport proteins

Summary For the purification of the bile acid transport proteins from rat hepatocytes high affinity taurocholic acid derivatives were synthesized. One type was designed for coupling to a Sepharose matrix and the other for copolymerization with acrylamide. The resulting matrices were found to be suitable for affinity chromatography and affinity electrophoresis. In both syntheses, 3, 12-dihydroxy-7-amino (tauro) cholic acid derivatives were used as the starting material, since substitution in this position had previously been shown not to alter transport affinity constant.     

Fluorescence detection and quantitation of recombinant proteins containing oligohistidine tag sequences directly in sodium dodecyl sulfate-polyacrylamide gels

Two fluorophore-nitrilotriacetic acid conjugates, Pro-Q Sapphire 365 and Pro-Q Sapphire 488 oligohistidine gel stains, have been developed for the fluorescence detection of fusion proteins containing oligohistidine tags directly in sodium dodecyl sulfate polyacrylamide gels, without the requirement for electroblotting, reporter enzymes or secondary detection reagents. Pro-Q Sapphire 365 oligohistidine gel stain exhibits bright-blue fluorescence (emission maximum = 450 nm) when illuminated with UV-A or UV-B light from a standard ultraviolet transilluminator. Pro-Q Sapphire 488 oligohistidine gel stain exhibits bright-green fluorescence (emission maximum = 515 nm) when illuminated with visible light from a laser-based gel scanner equipped with a 470 nm second-harmonic generation (SHG) or 488 nm argon-ion laser source. Typically, 25-65 ng of oligohistidine-tagged fusion protein in whole cell lysates is detectable using either stain. After documenting the fluorescence signal from the Pro-Q Sapphire dyes, gels may be post-stained with the red-fluorescent SYPRO Ruby protein gel stain in order to reveal the total protein pattern.