The use of dye-ligand affinity chromatography for the purification of non-enzymatic human plasma proteins

Literature data are analysed in this review on the use of immobilized triazine dyes for the characterization, isolation and purification of non-enzymatic human plasma proteins in both conventional and high-pressure liquid chromatography systems. Attention is focused on the mode of interaction between the dyes and these proteins, as well as on the advantages over previously reported techniques. Future developments are discussed.     

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

The use of recombinant fusion proteases in the affinity purification of recombinant proteins

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.     

Partial purification of rat liver glucocorticoid binding proteins by affinity chromatography

Dexamethasone (9-fluoro-16α-methyl-116,17,21-trihydroxy-1,4-pregnadiene-3, 20-dione) binding proteins from rat liver cytosol were purified approximately 6470 fold by the use of an affinity column in which deoxycorticosterone was linked to CH-Sepharose 4B through a disulfide linkage. The receptor proteins were eluted from the column by washing with β-mercaptoethanol. A preliminary Sephadex G-200 filtration step of the cytosol was necessary in order to separate the dexamethasone binding proteins from other glucocorticoid receptors.     

Rapid purification of the estrogen receptor by sequence-specific DNA affinity chromatography

Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.     

The purification of Aeromonas aminopeptidase by affinity chromatography

A competitive inhibitor for Aeromonas aminopeptidase has been prepared from the bromomethyl ketone derived from t-butyloxycarbonyl-l-leucine and successfully coupled to aminomethyl cellulose to form an adsorbent for affinity chromatography. The blocked form of the inhibitor was coupled to aminomethyl cellulose and then deblocked in aqueous trifluoroacetic acid to yield an insolubilized analog of an NH₂-terminal l-leucyl residue. This material was effective in binding the aminopeptidase and separating it from a contaminating endopeptidase, which has a similar isoelectric point and size. Separation of the aminopeptidase and endopeptidase was shown to be due to the specificity of the affinity adsorbent for the aminopeptidase, inasmuch as separation of the enzymes did not occur on a typical anion exchange column or on a hydrophobic column lacking a free amino group.     

Affinity chromatography in the industrial purification of plasma proteins for therapeutic use.

Affinity chromatography is a powerful technique for the purification of many proteins in human plasma. Applications cover the isolation of proteins for research purposes but also, to a large extent, for the production of therapeutic products. In industrial plasma fractionation, affinity chromatography has been found to be particularly advantageous for fine and rapid capture of plasma proteins from industrial plasma fractions pre-purified by ethanol fractionation or by ion-exchange chromatography. To date, affinity chromatography is being used in the production of various licensed therapeutic plasma products, such as the concentrates of Factor VIII, Factor IX, von Willebrand Factor, Protein C, Antithrombin III, and Factor XI. Most commonly used ligands are heparin, gelatin, murine antibodies, and, to a lesser extent, Cu(2+). Possible development of the use of affinity chromatography in industrial plasma fractionation should be associated to the current development of phage display and combinatorial chemistry. Both approaches may lead to the development of tailor-made synthetic ligands that would allow implementation of protein capture technology, providing improved productivity and yield for plasma products.     

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.     

Identification and purification of resveratrol targeting proteins using immobilized resveratrol affinity chromatography

The phytochemical resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring polyphenol with a plethora of health-beneficial properties, including a preventive role in cancer. We surmise that resveratrol may exert its diverse biological effects by interacting with specific target proteins, denoted RTPs. To test this possibility, resveratrol was immobilized on epoxy-activated agarose forming a resveratrol affinity column (RAC), which was used to detect and isolate RTPs. Distinct RTPs can be resolved on RAC by fractionation with increasing NaCl, followed by 1 mM ATP, and finally, with 1-2 mM resveratrol. A 22-kDa polypeptide, RTP-22, eluted with resveratrol was identified by MALDI-TOF MS and cloning/expression in Escherichia coli, as dihydronicotinamide riboside quinone reductase 2 (NQO2). The utility of RAC was additionally explored with extracts derived from different staging prostate cancer cells. NQO2 was most abundant in CWR22Rv1, a model for prostate cancer transition from androgen-dependent to the hormone-refractory state, but was marginally expressed in JCA-1 cells as representing more advanced stage prostate cancer. These results provide evidence for the existence of distinctive RTPs in mammalian cells and that RAC is a facile approach to identify and purify RTPs.     

Designer dyes: 'biomimetic' ligands for the purification of pharmaceutical proteins by affinity chromatography.

Affinity chromatography has been extensively refined over the past few years to meet the more stringent criteria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in process-scale protein purification. Exploitation of sophisticated molecular modelling techniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective 'biomimetic' ligands for the target proteins.     

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification.     

Estrogen receptor purification by affinity chromatography using an orange triazine dye

A rapid two-step procedure was devised for the purification of the estrogen receptor from the calf uterus. A 900- to 1700-fold purification of the estrogen receptor was obtained using ammonium sulfate precipitation followed by dye affinity chromatography with Reactive Orange 14 immobilized to Sepharose. The Reactive Orange 14-Sepharose was used to purify the estrogen receptor in the presence or absence of estradiol as well as to purify the progesterone receptor. The purified estrogen receptor retained its estradiol- and DNA-binding properties and sedimented into sucrose gradients as the 5 S receptor dimer. The Reactive Orange 14-Sepharose is easily prepared and offers a higher yield and purity of the estrogen receptor than that afforded by estrogen- or heparin-Sepharose chromatography.