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.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.     

DNA purification by triple-helix affinity precipitation

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.     

Precipitation of RNA impurities with high salt in a plasmid DNA purification process: use of experimental design to determine reaction conditions

The use of high salt solution to precipitate RNA in a pharmaceutical-grade plasmid DNA purification process was investigated. Five antichaotropic salts were tested for their potential to precipitate RNA. Calcium chloride was by far the best precipitant with high RNA removal in a very short incubation time. Calcium chloride precipitation conditions were investigated at two stages of a plasmid purification process using experimental design techniques. The effect of up to five factors on RNA precipitation and plasmid recovery was assessed by statistical modeling. Optimized conditions for calcium chloride precipitation were then introduced to the plasmid purification process resulting in the efficient removal of most impurities (RNA, chromosomal DNA, proteins, and endotoxins).     

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.     

Fractional precipitation of plasmid DNA from lysate by CTAB

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.     

Single step immobilized metal ion affinity precipitation/chromatography based procedures for purification of concanavalin A and Cajanus cajan mannose-specific lectin

Concanavalin A and a mannose-specific lectin could be precipitated specifically from extracts of jack bean and Cajanus cajan seeds, respectively, using metal charged EGTA. Single step purification of the lectins was also possible using iminodiacetic acid-Sepharose charged with metal ions. Nondenaturing electrophoresis in polyacrylamide gel and that performed in presence of SDS ascertained homogeneity of the isolated lectins. The migration behavior of the purified lectins was comparable with those of the lectins purified using alternative procedures.     

Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers

Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments.     

Metal affinity protein precipitation: effects of mixing, protein concentration, and modifiers on protein fractionation

Previous work by us and others has shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, we probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation as our model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control the effects contacting conditions on precipitate yield, purity, and particle size distribution. First, we studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, we examined the role of the molecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine gamma-globulins, carried out at a nominal 1-L scale; glycine was examined as a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle size. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations. (c) 1995 John Wiley & Sons, Inc.     

His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Di(2‐ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X‐45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R₂. The Ni(II)‐chelate reverse micelles were characterized for the purification of recombinant hexahistidine‐tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R₂ was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni²⁺ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two‐stage purification by the metal‐chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3/lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOS and lacO3 were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO1 and 1-1.2 x 10(-10) M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.     

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.     

Single-step purification of a small non-mAb biologic by peptide-ELP-based affinity precipitation

Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a K_D of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation.     

Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process

Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.     

One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.     

Immobilization of oligonucleotides on a large pore support for plasmid purification by triplex affinity interaction

Triplex affinity interaction provides a new process for the purification of plasmid DNA, which is especially suited to meet the demands of a gene therapy use. We developed a method for the functionalization of a large pore affinity support suitable for this application. A 5-modified DNA oligonucleotide containing an aldehyde group was coupled to adipic acid hydrazide functionalized Sephacryl beads with a yield of 31% (over all immobilization yield 22.6% from starting oligonucleotide). The resulting selective and covalent immobilization of the ligand via a 16 atom, hydrophilic linker arm enables the oligonucleotide bases to freely bind to the target sequence. The proposed method provides affinity supports that might be used in large scale affinity purification of plasmid DNA.This revised version was published online in October 2005 with corrections to the Cover Date.     

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with impractically low DNA yields. We have optimized the procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 microg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.     

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.     

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.