Biomedical subjects

G Wellnhofer

Publications and source records attributed to G Wellnhofer.

4 recordsLinked to original sources

PubMed2000

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes.

Amino Acid Sequence↗

PubMed1999

Selection of a peptide ligand to the p75 neurotrophin receptor death domain and determination of its binding sites by NMR.

The p75 neurotrophin receptor (p75(NTR)) contains a conserved death domain module similar to that of the cytotoxic receptors Fas and TNFR-1. Here, we describe the selection of peptide ligands from a combinatorial library using a variation of the selectively-infective phage (SIP) method directed to the death domain of p75(NTR). The binding sites on the death domain of p75(NTR) were identified for a 15 amino acid residue peptide by nuclear magnetic resonance (NMR) spectroscopy. The selected peptides may be useful for probing the function of the p75(NTR) death domain and aid in defining its downstream signalling mechanism.

Amino Acid Sequence↗

PubMed1996

High affinity endotoxin-binding and neutralizing peptides based on the crystal structure of recombinant Limulus anti-lipopolysaccharide factor.

Lipid A, the conserved portion of endotoxin or lipopolysaccharide, is the major mediator of septic shock, and therefore endotoxin-neutralizing molecules could have important clinical applications. The crystal structure of recombinant Limulus anti-lipopolysaccharide factor (rLALF) (Hoess, A., Watson, S., Siber, G. R., and Liddington, R. (1993) EMBO J. 12, 3351-3356), has been used to design synthetic peptides comprising different parts of the exposed amphipathic loop in the proposed endotoxin-binding domain of rLALF. We investigated the minimal requirements of rLALF for endotoxin and lipid A binding with linear 10-mer peptides. Only one linear peptide, corresponding to amino acids 36-45 of rLALF, was able to bind lipid A and endotoxin above background levels. Cyclic peptides, however, bind lipid A and endotoxin with high affinity, presumably by mimicking the three dimensional characteristics of the exposed hairpin loop. The cyclic peptide including amino acids 36-47, LALF-14, has a lipid A binding activity comparable to the high affinity endotoxin-binding peptide polymyxin B. LALF-14 has an improved serum half-life compared with its linear counterpart, and it is not toxic for cultured human monocytes or red blood cells. In mice, it blocks tumor necrosis factor-alpha induction after endotoxin challenge. The characterization of the minimal endotoxin-binding domain of rLALF and, importantly, its structure provided a basis for designing small molecules that could have prophylactic and/or therapeutic properties in humans for the management of septic shock.

Animals↗

PubMed1994

Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.

Trinucleotide phosphoramidites representing codons for all 20 amino acids have been prepared and used in automated, solid-phase DNA synthesis. In contrast to an earlier report, we show that these substances can be used to introduce entire codons into oligonucleotides in excess of 98% yield, and are ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.

Base Sequence↗