Arne Skerra

Sequence analysis and bacterial production of the anti-c-myc antibody 9E10: the VH domain has an extended CDR-H3 and exhibits unusual solubility

The cDNAs for the two variable domains of the antibody 9E10 were cloned from the hybridoma cell line. A chimeric 9E10 Fab fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional Fab fragment was isolated in a single step via a His6‐tag, which also served for its recognition by a nickel chelate‐alkaline phosphatase conjugate. Thus, the recombinant Fab fragment permitted the immunochemical detection of the myc tag in a sandwich ELISA. The dissociation constant for the interaction with the myc tag peptide was determined as 80±5 nM by fluorescence titration. In an attempt to produce the smaller 9E10 Fv fragment it was found that its VH domain alone can be readily isolated from E. coli as a soluble protein. This unusual behaviour may be explained by the 18 amino acid‐long CDR‐H3 and could be of value in the design of `single domain antibodies.

A Zn(II)-binding site engineered into retinol-binding protein exhibits metal-ion specificity and allows highly efficient affinity purification with a newly designed metal ligand

BACKGROUNDnThe Zn(II)-binding site from the active center of human carbonic anhydrase II, formed by three His side chains, can be grafted onto the recombinant serum retinol-binding protein (RBP). The artificial binding site in the resulting variant RBP/H3(A) has high affinity for Zn(II) and stabilizes the protein against denaturation.nnnRESULTSnThe metal-ion specificity of the grafted Zn(II) binding site in RBP/H3(A) was investigated. Both Cu(II) and Ni(II) bound with high affinity, although the Kd values were not as low as for Zn(II) binding. Competition experiments with the chelate ligands iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) suggested that both Ni(II) and Cu(II) bound to the protein in an octahedral manner with three vacant coordination sites, as previously observed for Zn(II). A substituted pyrrolidine-dicarboxylic acid was designed as a structurally rigid IDA compound and coupled to a matrix. Using this support in an immobilized metal affinity chromatography (IMAC), RBP/H3(A) was purified from the bacterial cell extract in one step with unprecedented efficiency.nnnCONCLUSIONSnAlthough the His3 metal-binding site used here had been removed from the substrate pocket of an enzyme and exposed to solvent on a protein surface, it showed clear selectivity for Zn(II) compared to Cu(II) and Ni(II). Thus the properties of this structurally defined metal-binding site (which are not shared by isolated His residues or flexible oligo-His tags) can be preserved when it is added to proteins. An IMAC matrix with improved behaviour was designed, allowing highly selective purification of RBP/H3(A) and of His6-tagged RBP as well. Such rational design of supramolecular recognition may be generally useful in the fields of protein engineering and drug design.

Structure and Dynamics of One Dimensional Ionic Solutions in Narrow Transmembrane Channels — A Molecular Dynamics Simulation Study —

Molecular dynamics studies for the voltage driven transport of the alkali metal ions Li+, Na+ and K+ through gramicidin A-type channels in the presence of water molecules are presented. It is demonstrated that the number of water molecules in the channel plays a key role for the cation selectivity.