Erich Hochuli

New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues

A novel nitrilotriacetic acid adsorbent has been prepared for metal chelate affinity chromatography. The new resin is a quadridentate chelate former and specially suitable for metal ions with coordination numbers of six, since two valencies remain for the reversible binding of biopolymers. It has been found to chelate Cu2+ and Ni2+ strongly and to be superior to the known iminodiacetic acid adsorbent. Charged with Ni2+, it was evaluated for the ability to bind peptides and proteins containing neighbouring histidine residues. The remarkable specificity found makes it an attractive addition to the range of adsorbents for metal chelate affinity chromatography.

Large-scale chromatography of recombinant proteins

When recombinant proteins are expressed in bacterial cells and subsequently grown in fermentation tanks, there remains the problem of recovering the product in pure form. The empirical knowledge gained upon recovery of recombinant proteins indicates that a one-step purification process is very unlikely to succeed. However, combinations of modern techniques, such as immunoaffinity chromatography or immobilized-metal affinity chromatography, with classical techniques, such as ion-exchange chromatography, seem to be suitable for large-scale recovery of recombinant proteins.

Immobilization of monoclonal antibodies for affinity chromatography using a chelating peptide

A procedure was developed for oriented immobilization of monoclonal antibodies on a solid support. The technique involves the specific oligosaccharide-directed covalent modification of the monoclonal antibody (mAb) with the chelating peptide, Lys-Gly-(His)6, in conjunction with immobilized metal ion affinity chromatography. Chelating peptide-mAb conjugates with a molar ratio of 2.2 retained full antigen binding activity. On immobilization of the modified antibodies on a nickel affinity resin, the molar antigen binding ratio was 1.4. The high antigen binding capacity is indicative of oriented immobilization providing maximum access for the antigen. The described method can be used for the preparation of high-capacity immunosorbents for affinity chromatography and it is applicable for all immunoglobulin classes.

Interaction of hexahistidine fusion proteins with nitrilotriacetic acid-chelated Ni2+ ions

The strong interaction of hexahistidine fusion proteins with nitrilotriacetic acid-chelated Ni2+ ions was explored in three different applications. The interaction has been used successfully for the purification of recombinant proteins by immobilized metal ion affinity chromatography. In addition, it was used to immobilize recombinant enzymes to the nitrilotriacetic acid resin charged with Ni2+ ions for application in organic chemistry. Furthermore, it was used in an analytical method to identify hexahistidine fusion proteins. A nitrilotriacetic acid derivative was labeled with biotin. This reagent was utilized to trace hexahistidine fusion proteins spiked with Ni2+ ions. The biotin was detected with streptavidin-labeled alkaline phosphatase. Through the addition of the soluble substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride, the alkaline phosphatase catalyzes the formation of an intense, insoluble dark-purple precipitate.

Specificity of the immunoadsorbent used for large-scale recovery of interferon α-2a

Abstract After immunoaffinity chromatography, interferon α-2a synthesized in bacteria is not homogeneous. Beside oligomers and monomers, a fragment of molecular weight 15 000 was observed. Amino acid analysis and the determination of the amino terminal amino acid sequence of the fragment indicate that this polypeptide represents an interferon α-2a molecule lacking the 22 terminal amino acids. In order to define the epitope on the interferon α-2a molecule recognized by the immunoadsorbent, the binding to the immunoadsorbent of interferon α-2a fragments prepared by cyanogen bromide cleavage was studied. The results suggest that cyanogen bromide fragments Arg 22—Met 59 and Lys 112—Met 148 are recognized. Since the oligomers, the monomers and the fragment of molecular weight 15 000 of interferon α-2a share these sequences, the different forms are as expected co-purified on the immunoadsorbent.

Method to identify hexahistidine fusion proteins in crude cellular lysate

The strong interaction of chelated nickel ions with a hexahistidine peptide was utilized to identify hexahistidine fusion proteins in crude cellular lysates. The technique involves a Ni2+-nitrilotriacetic acid derivative labelled with alkaline phosphatase. The new reagent was used to probe for hexahistidine fusion proteins after SDS-PAGE electrophoresis and blotting onto nitrocellulose. The minimal amount of fusion protein which could be detected with this method was 0.1 μg.