Affinity ligands for industrial protein purification

Significant efforts are put into the design of large-scale purification processes of proteins due to great demands regarding cost efficiency and safety. In order to design an effective purification scheme the unit operations need to be reduced to a minimum. In this review we are discussing proteinaceous ligands as well as small synthetic mimics for use in affinity chromatography for large-scale applications. Different advantages as well as drawbacks of the two approaches are outlined.     

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.     

Affinity purification methods. Improved procedures for cyanogen bromide reaction on agarose.

The high solubility of cyanogen bromide inN-methyl-2-pyrrolidone has afforded an efficient means of controlling the amount of the reagent used to activate an agarose preparation. By controlling the activation step, one can determine the final concentration of the leash moiety. The upper limits of ε-aminocaproic acid incorporation seem to be about 5, 20, and 60 μequiv/ml gel, respectively, for 2, 4, and 6% agarose gels. It is important to conduct the cyanogen bromide reaction at 4–10°C, but the coupling step may be done at room temperature.     

Affinity chromatographic purification of papain.

The reinvestigation of the affinity chromatographic method of purifying papain has been carried out. It has been reported that papain could be purified by taking advantage of the affinity of the enzyme for the insolubilized peptide inhibitor, agarose-Gly-Gly-Tyr(Bz)-Arg. Using pure tetrapeptide obtained commercially and standard coupling procedures, a significant purification of papain could not be achieved. Both active and nonactivatible enzyme bound to a column prepared in this manner were eluted together by the use of deionized water. An affinity medium with properties similar to those reported by Blumberg et al. was obtained by removal of the benzyl group on tyrosine prior to coupling with agarose. The deprotected tetrapeptide was also synthesized by an independent route and inhibition constants for the binding of the protected and deprotected tetrapeptide to papain were determined in kinetic experiments.     

Affinity chromatographic purification of morphine antibody.

Morphine-6-hemisuccinate was synthesized and linked to agarose affinity beads by either direct amide bond formation or by an N-hydroxysuccinimide ester intermediate using various conditions. The various preparative routes resulted in differing ampunts of covalently bound ligand. Affinity chromatography of morphine antisera with a variety of eluting solvents indicated that 0.5 M acetic acid and 1 M propionic acid were most efficacious for eluting the bound antibody. Affinity isolation of a papain digest of purified antibody yielded fragments with reactivity and other characteristics consistent with their being designated as morphine antibody Fab fragments.     

Affinity purification of hexosaminidases

Hexosaminidases A and B were purified by affinity chromatography from normal gastric mucosa, after preliminary separation of isozymes by anion exchange chromatography. Heparin and mannosamine were coupled to Sepharose 4B and used as affinity matrices and the purified enzymes were found to be homogeneous when analysed by polyacrylamide slab gel electrophoresis. This combination of 2 novel affinity chromatographic procedures is superior to existing methods in that a final yield of over 70% could be achieved. Also, the number of steps required to obtain homogeneity are less in contrast to the conventional methods used previously.     

Affinity purification of aminoacyl-tRNA

A procedure for separating Escherichia coli aminoacyl-tRNA from unacylated tRNA or components of the aminoacylation reaction, thereby achieving an aminoacyl-tRNA product with a very high specific activity, is described. The method utilizes the specific recognition of aminoacyl-tRNA for E. coli protein synthesis elongation factor Tu which has been immobilized on an affinity matrix. The application of the affinity procedure as a means of purifying a single aminoacyl-tRNA from an unfractionated mixture of tRNAs is also discussed.     

Affinity chromatography system for parallel purification of recombinant protein samples

In protein engineering, the tasks of generating and testing a large number of variants of a molecule and of optimizing expression conditions for one distinct molecule create the need for purification methods that can handle a large number of samples simultaneously. We describe the development and some application results of a simple affinity chromatography system that can be used for the parallel purification of 24 protein samples, yielding sufficient quantities for biochemical and functional analysis. Advantages of this system over existing systems are as follows. Compared with commercially available complete chromatography systems, the costs of this system are minimal. In comparison with vacuum systems with various outlets, and with batch purification systems where centrifugation is necessary, this system allows gentler processing of the samples. This could be important for proteins that are easily damaged.     

Efficient purification of PCR products using ultrafiltration.

The use of disposable ultrafiltration devices for the purification of PCR-amplified DNA products from the other components of the reaction mixture is outlined. The advantages of using a 100,000 molecular weight cutoff membrane are discussed. The integrity of the membrane-purified DNA is checked by its successful use in sequencing, ligation and cloning procedures.     

New methods for the purification of vertebrate vitellogenin.

Two procedures are described for the isolation of vitellogenin from plasma. Vitellogenin can be selectively precipitated from the plasma of estrogen-treated animals by Mg²⁺ in the presence of EDTA. The vitellogenin thus obtained is >99% pure and is relatively undegraded. In the second procedure, vitellogenin can also be isolated by DEAE-cellulose chromatography, resulting in preparations that contain <2% albumin. In combination, these two isolation procedures yield vitellogenin preparations of very high purity.     

Affinity purification of an interferon-induced human guanylate-binding protein and its characterization

Among the proteins that are synthesized only in interferon-treated human cells, a Mr = 67,000 protein has been previously identified by its binding to guanylate agaroses. After a 24-h treatment of human diploid fibroblasts with 200 units/ml of interferon-gamma, about 3 X 10(5) molecules of guanylate-binding protein (GBP) accumulate in each cell. We have developed a one-step purification procedures for GBP using guanylate affinity chromatography. To further elucidate the specific binding of this protein to guanylates, we have used a photoactive probe, 8-azidoguanosine [alpha 32P] triphosphate for the labeling of the GBP. Photolysis of the 8-azido-[alpha-32P]GTP in the presence of GBP results in the covalent attachment of the 32P-guanylate to the GBP. This photolabeling reaction can be inhibited only by guanylates but cannot be inhibited by other nucleotides, suggesting a specific association of GBP to guanylates. Using the purified GBP as an immunogen, we have successfully made rabbit antiserum for GBP. Both the GBP antigen and its guanylate-binding activity are detected only in the cytoplasm of interferon-treated human fibroblasts. The synthesis of the mRNA of GBP is also found in mice exposed to endogenous interferon and in interferon-treated human lymphocytes.     

Affinity purification of lipid vesicles

We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 x 10(13) active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 x 10(8) M(-1). We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format.     

Affinity purification of histidine-tagged proteins

Expression of recombinant proteins is a standard technique in molecular biology and a wide variety of prokaryotic as well as eukaryotic expression systems are currently in use. A limiting step is often the purification of the expressed recombinant protein, particularly if mammalian expression systems that yield low expression levels are employed. Here, we discuss the advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni²⁺-NTA chromatography.Abbreviations GST glutathione S-transferase - NTA nitrilotriacetic acid - His histidine - PAGE polyacrylamide gel electrophoresis     

Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B.

A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.     

Affinity chromatographic purification of beta-glucosidase of Candida gulliermondii.

A beta-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethyleneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacrylamide disc electrophoresis of the purified enzyme gave a single band. A km of 1.25 x 10(-4)M was obtained using p-nitrophenyl beta-D-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at temperature of 37 degrees C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48,000 as estimated by sucrose density gradient centrifugation.     

Affinity purification of the photoreceptor cGMP-gated cation channel.

The cGMP-gated cation channel is a member of a new family of channel proteins that appear to be directly regulated by cyclic nucleotides. A protein with a subunit molecular mass of 78 kDa that exhibits cGMP-gated calcium flux when reconstituted into phospholipid-containing vesicles has been purified using 8-bromo-cGMP-agarose affinity chromatography. This channel activity is sensitive to the inhibitor l-cis-diltiazem. Treatment of the reconstituted channel with trypsin abolishes the l-cis-diltiazem sensitivity. Apparent endogenous proteolysis can also result in smaller molecular weight polypeptides that exhibit cGMP-gated channel activity but are insensitive to l-cis-diltiazem. These results show that the channel can bind cGMP and that it contains a l-cis-diltiazem inhibitory domain that is distinct from the cGMP-binding domain.     

Affinity purification of specific DNA fragments using a lac repressor fusion protein

A new method for purification of specific DNA sequences using a solid phase technique has been developed based on a fusion between the Escherichia coli lac repressor gene (lacI) and the staphylococcal protein A gene (spa). The fusion protein, expressed in Escherichia coli, is active both in vivo and in vitro with respect to its three functional activities (DNA binding, IPTG induction, and IgG binding). The recombinant protein can be immobilized in a one-step procedure with high yield and purity using the specific interaction between protein A and the Fc-part of immunoglobulin G. The immobilized repressor can thereafter be used for affinity purification of specific DNA fragments containing the lac operator (lacO) sequence.     

Affinity chromatography purification of diphtheria toxin

NAD was covalently linked to Sepharose-4B using a 6 carbon spacer. Sterile, dialyzed spent culture medium containing 100 Lf/ml of diphtheria toxin or material concentrated by (NH₄)₂SO₄ precipitation containing 1500 Lf/ml, was chromatographed on a column of NAD–Sepharose. Ultraviolet absorbing material which did not flocculate with diphtheria antitoxin was eluted with 0.02M phosphate buffer. When the elation buffer was changed to one containing 0.5M NaCl, purified toxin was eluted off the column.