Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli

In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a β-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn²⁺-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml⁻¹ PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher β-glucanase concentration (65.6 kU ml⁻¹), i.e. 1.5-fold compared to the internal adsorbent system. An erratum to this article can be found at     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Expression and affinity purification of recombinant proteins from plants

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.     

Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography

The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.     

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.     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

Single-process expression and purification of multiple recombinant proteins through cocultivation and affinity purification

Multiple recombinant proteins can be expressed simultaneously by inoculating multiple seed cultures into a single growth medium and inducing protein expression at a single time point. Up to three recombinant proteins can be individually purified from such a mixed culture (cocultivation) through the use of a combination of a multihistidine and a modified intein as affinity tags and the Ni sepharose and chitin as affinity matrices. This method may facilitate the study of protein complexes by rapidly obtaining multiple protein components in a single process and may potentially increase the efficiency of recombinant protein production at research and industrial scales.     

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.     

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

Subunit affinity chromatography and its application to the isolation of aryl sulfatase A enzymes

The monomeric form of rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) was covalently coupled to CNBr-activated Sepharose and the catalytic properties of the covalently coupled monomer subunit were examined. The immobilized subunit showed one pH optimum near pH 5.6 which appears to be the characteristic pH optimum of the monomer. The enzyme-Sepharose complex exhibited the characteristic anomalous kinetic behavior at pH 5.5 but there was no turnover-induced inactivation of the immobilized enzyme at pH 4.5. The covalently coupled subunit column was examined for its ability to act as a subunit affinity chromatography medium. It was found that dissolved aryl sulfatase A was removed from solution at pH 4.5 and pH 5.0, I = 0.2, and became associated with the affinity column of Sepharose-aryl sulfatase A. The retained subunit of the enzyme could subsequently be quantitatively eluted with 0.2 m Tris-HCl, pH 7.5. Extraneous protein such as bovine serum albumin did not measureably affect the rate or equilibrium for association of the enzyme to the covalently bound subunit. The extent of binding of the enzyme to the affinity column was found to be strongly dependent on the time of equilibration and on the pH. About 90% of the enzyme was retained after 24 h at pH 5.0, I = 0.2. Under otherwise comparable conditions, use of Sepharose-6MB resulted in slightly faster association than did Sepharose-4B. Under the experimental conditions employed, the total capacity of the affinity column was approx 50% of the total aryl sulfatase A coupled to the Sepharose. The rabbit liver subunit column also permits the purification of several other aryl sulfatase A enzymes. Thus, the subunit affinity column provides a simple, convenient, and rapid procedure for the isolation of most mammalian aryl sulfatase A enzymes as well as for studying inter- and intraspecific subunit association interactions.     

Application of affinity chromatography to the purification of collagenase for the isolation of insoluble elastin.

ClostrIdium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.19) was purified by batchwise separation with DEAE-cellulose followed by affinity chromatography on a column of alkali-treated elastin. The N-terminal amino acid profile of elastin isolated from bovine ligamentum nuchae using this enzyme preparation was compared with that of a duplicate sample purified with a mixture of collagenase I and II (Yoshida, E, and Noda, H. (1965) Biochim. Biopsys. Acta 105, 562-574). An approx. three-fold decrease in the molar concentration of N-terminal residues and a considerable reduction in their number was obtained by using the former enzyme preparation.     

Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase

This protocol describes affinity purification of bacterially expressed, recombinant membrane proteins fused with calmodulin-binding domains. As exemplified by the Escherichia coli nicotinamide nucleotide transhydrogenase, this method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA and, unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). Our protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product.     

Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.