Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent

Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components. We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products. In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins. Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed. Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents.     

Factors affecting endotoxin removal from recombinant therapeutic proteins by anion exchange chromatography

Removal of endotoxins from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of the bacterial expression systems widely used to manufacture therapeutic proteins. In this study we investigated various parameters affecting anion exchange chromatography to selectively remove endotoxins from therapeutic proteins. NY-ESO-1, Melan-A, and SSX-2 are different recombinant proteins used in this study, all of them are cancer antigens currently developed as potential immunotherapeutic agents. We found that by using a commercially available Q XL resin in a flow-through mode, endotoxin could be effectively removed from these proteins while maintaining very acceptable protein yields. The ratio of resin volume to endotoxin load was analyzed to determine the endotoxin binding capacity of the resin. In our hands at least 900,000 endotoxin units (EU) could be loaded per ml of Q XL resin. Solution conductivity could be increased to 20 mS/cm to minimize protein loss by weakening protein-resin attraction, and pH could be increased to enhance endotoxin removal by weakening endotoxin-protein attraction. Endotoxin levels were ultimately decreased to below 0.5 EU per microg of protein, an over 2000-fold reduction in this single step. A successful scale-up of these processes in which column volume was increased 100-fold was performed under cGMP conditions with over 80% protein recovery.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins’ application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.     

Specific binding of endotoxin to human monocytes and mouse macrophages: serum requirement

Specific binding of Bordetella pertussis and Neisseria meningitidis endotoxins to human monocytes and murine macrophages was demonstrated. Binding of B. pertussis endotoxin could be inhibited by endotoxins of Salmonella minnesota, Escherichia coli, and Klebsiella pneumoniae, the extent of inhibition being dependent on the origin of the lipopolysaccharides and on the origin of the mononuclear phagocytic cells. The binding of B. pertussis and N. meningitidis endotoxins which was mediated by the polysaccharide region of the endotoxins was serum dependent. The results indicated that the binding of endotoxin was promoted neither by natural antibodies directed against the endotoxin nor by proteins known to combine with endotoxins: immunoglobulins, albumin, or fibronectin; we have provided some evidence that complement components may play a role in the specific binding of endotoxins to the monocyte/macrophage membrane.     

Endotoxin removal from protein solutions

Endotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses. Because of their high toxicity in vivo and in vitro, their removal is essential for a safe parenteral administration. A general method for the removal of endotoxins from protein solutions is not available. Methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. Various techniques described in the patent literature are not broadly applicable, as they are tailored to meet specific product requirements. Besides ion-exchangers and two-phase extraction, affinity techniques are applied with varying success. Also, taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose. After giving an overview of the properties of endotoxins and the significance of endotoxin contamination, this review intends to provide an overall picture of the various methods employed for their removal. Avenues are pointed out how to optimise a method with regard to the specific properties of endotoxins in aqueous solution.     

Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product.

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Removal of tightly bound endotoxin from biological products

The method for endotoxin removal described in this paper is useful for separation of tightly bound endotoxin from biological products, particularly those produced in Escherichia coli in the form of inclusion bodies for which a denaturation step is required to solubilise the product. We employed guanidine hydrochloride and ammonium sulphate in combination with hydrophobic interaction chromatography (HIC). These conditions enable binding of the endotoxin to the matrix, giving unbound product in the column flow-through. This makes the method generally applicable to biological products. An endotoxin reduction of about 3.7 logs was achieved; from as much as 1,100,000 EU mg(-1) in the solubilised material to about 200 EU mg(-1) in the product purified by this method. The method was developed for a cervical dysplasia vaccine, a fusion protein comprising L2, E7 and E6 from Human Papilloma Virus type 16, because both conventional and commercially available methods of endotoxin removal were ineffective in removing the tightly bound endotoxin from this product.     

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.     

Effect of lead acetate on the susceptibility of rats to bacterial endotoxins

Selye, H. (Université de Montréal, Montreal, Canada), B. Tuchweber, and L. Bertók. Effect of lead acetate on susceptibility of rats to bacterial endotoxins. J. Bacteriol. 91:884-890. 1966.-A single, normally well-tolerated, intravenous injection of lead acetate increases the sensitivity of the rat to the endotoxins of various gram-negative bacteria about 100,000 times above normal. Under the conditions of these experiments, the mortality and organ changes normally produced by the intravenous injection of 100 mug of Escherichia coli endotoxin were essentially the same as those obtained by use of 1 nanogram in lead-sensitized rats. The sensitizing effect of lead acetate for E. coli endotoxin is greatest when the two agents are given simultaneously. However, considerable sensitization is still detectable when endotoxin is injected up to 1 hr before or 7 hr after sensitization with lead. No sensitization was noted when the endotoxin was administered 24 hr before or after lead acetate. Under our experimental conditions, the minimal dose of lead acetate which could still induce significant sensitization to E. coli endotoxin was 1 mg per 100 g of body weight. Although lead acetate induces a high degree of susceptibility to various endotoxins, other reticuloendothelial blocking agents did not acquire unusual toxicity after pretreatment with lead. Finally, none of the other metals or reticuloendothelial blocking agents tested could duplicate the pronounced decrease in endotoxin resistance induced by lead acetate.     

Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association

This work combines two well-established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self-cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed. In this system, the PHB-binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB-binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self-cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries.     

Endotoxin removal by charge-modified filters.

The effects of positively charged nylon and depth (cellulose-diatomaceous earth) filters on endotoxin removal from various solutions were evaluated. The charged filter media removed significant amounts of Escherichia coli and natural endotoxin from tap water, distilled water, sugars, and NaCl solutions; no significant removal of endotoxin was observed with negatively charged filter media. The extent of removal was influenced by pH, the presence of salts, and organic matter. Such media may be useful for the control of endotoxins in raw-product water or solutions used to prepare parenteral drug products or in other fluids where endotoxin control is desired.     

Endotoxin removal by charge-modified filters

The effects of positively charged nylon and depth (cellulose-diatomaceous earth) filters on endotoxin removal from various solutions were evaluated. The charged filter media removed significant amounts of Escherichia coli and natural endotoxin from tap water, distilled water, sugars, and NaCl solutions; no significant removal of endotoxin was observed with negatively charged filter media. The extent of removal was influenced by pH, the presence of salts, and organic matter. Such media may be useful for the control of endotoxins in raw-product water or solutions used to prepare parenteral drug products or in other fluids where endotoxin control is desired.     

A simplified method for purification of recombinant soluble DnaA proteins

An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.     

Carcinoscorpius rotunda cauda amoebocyte lysate for detection of endotoxins--its preparation, stability, sensitivity and comparison with Limulus amoebocyte lysate

Carcinoscorpius amoebocyte lysate (CAL) was prepared from C. rotunda cauda by a modification of the method described by Mahalanabis et al. [Indian J Med Res, 70 (1979) 35]. Seasonal variation as well as batch variation was observed in the yield of haemolymph and the total lysate protein. In the presence of E. coli lipopolysaccharide (pure, free endotoxin) and E. coli and Salmonella cell suspensions (bound endotoxin), the CAL formed a gel after incubation at 37 degrees C. The gelling time varied from 10-90 min depending on the concentration of endotoxin used; higher concentrations formed gel more rapidly. The endotoxin detection capacity (sensitivity) of the lysate preparations was influenced by the season in which prepared, but not by the total protein content. Ten fold increase in the sensitivity was achieved by a purification step using chloroform. Although subsequent frozen storage with or without lyophilization did not alter the initial sensitivity, it was either decreased considerably or lost totally when the lysate was stored for 4 months at 4 degrees C or for 2 months at 30 degrees C. Under the same conditions, Limulus lysate was more stable. The lost sensitivity could not be regained by the incorporation of divalent cations (Ca2+ and Mg2+). The CAL preparations in general were able to detect as little as 10-100 pg of endotoxin or as few as 10(3) cells of E. coli or 10(4) cells of Salmonella and were comparable to LAL. CAL could be used successfully in lieu of Limulus amoebocyte lysate in the detection and assay of endotoxins.     

Determination of bacterial endotoxin admixtures in inactivated influenza vaccines

The materials substantiating the possibility of using the method for the determination of the lethal effect of endotoxin on dactinomycin-treated mice are presented. This determination is made with a view to detecting the admixtures of endotoxins in whole-virion and subvirion inactivated influenza vaccines at different stages of their manufacture, as well as in the final product. The proposed test is highly sensitive, rather simple in its practical realization and can be used for evaluating the degree of the purification of influenza vaccines from endotoxins.     

Tritiation of endotoxin.

Tritiated endotoxin was synthesized by three different methods: (1) sodium boro[3H]hydride reduction of native endotoxin; (2) sodium boro[3H]hydride reduction of endotoxin that had been oxidized previously with sodium metaperiodate; and (3) exposure of dry endotoxin to 3H2 gas. Sodium borohydride reduces aldehyde groups and sodium metaperiodate oxidizes vicinal glycol groups to aldehydes. Chromatographic analysis of the three tritiated endotoxins, using agarose, revealed that the biological activity associated with each labeled product appeared at the void volume, and in each case the biological activity coincided with a peak in radioactivity. The labeled product of the first method had a specific radioactivity of 0.18 mCi/g and a biological activity equal to that of native endotoxin. The labeled products of the second and third methods had specific activities of 2.1 mCi/g and 60.0 mCi/g, respectively, while their biological activities were one hundred-fold less than native endotoxin, as determined by the Limulus amebocyte lysate assay. These three labeled endotoxins are potentially ueled endotoxin.     

3种革兰氏阴性细菌及其L型内毒素含量的测定

本文采用鲎试剂对大肠杆菌（ＡＴＣＣ２５９２２）、伤寒杆菌、绿脓杆菌（ＡＴＣＣ７８５３）及它们的Ｌ型内毒素的含量进行测定。结果显示细菌型与细菌Ｌ型均具有内毒素,但细菌Ｌ型内毒素含量较细菌型低（约为１／３￣１／２）。因此,认为细菌Ｌ型仍有一定的致病性。     

Protein concentration effect on protein-lipopolysaccharide (LPS) binding and endotoxin removal

It is known some proteins can disaggregate endotoxins and form complexes with lipopolysaccharide (LPS). Nevertheless, how protein concentration affects protein-LPS binding and endotoxin removal is unknown. In this study, protein samples at various concentrations were incubated with endotoxin samples at a fixed concentration. The mixtures were filtered by ultrafiltration membranes. As protein concentration increased, the amount of endotoxin detected in the filtrates increased too. This result indicates protein concentration has significant effect on protein-LPS binding and the amount of endotoxin disaggregated.