Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21.

Global patterns of human DNA sequence variation (haplotypes) defined by common single nucleotide polymorphisms (SNPs) have important implications for identifying disease associations and human traits. We have used high-density oligonucleotide arrays, in combination with somatic cell genetics, to identify a large fraction of all common human chromosome 21 SNPs and to directly observe the haplotype structure defined by these SNPs. This structure reveals blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes.

Isolation of erythropoietin receptor agonist peptides using evolved phage libraries.

Cyclic peptides capable of activating the erythropoietin receptor (EPOR) were isolated from phage display libraries by screening with a novel EPOR-IgG fusion protein reagent. A parental clone ERB1 (EPO Receptor Binder 1) was first isolated from a phage display library displaying 38 random amino acids as an N-terminal fusion with the M13 minor capsid protein, pill. An evolved library was then produced from the parental sequence using an oligonucleotide saturation mutagenesis strategy which yielded EPOR binding sequences with 20 times the relative affinity of ERB1. Two synthetic peptides were constructed from these sequences both of which bind the EPO receptor in specific ELISA, and act as full agonists in EPO dependent cell proliferation assays. These peptides are 18 amino acids in length, disulfide-bonded, and have a minimum consensus sequence of CXXGWVGXCXXW, where X represents positions tolerant of several amino acids.

Expression and ligand binding assays of soluble cytokine receptor-immunoglobulin fusion proteins.

We have developed a cloning vector for the expression of type I cytokine receptor, NO, extracellular domain (ECD)-mouse IgG1 Fc fusion proteins. The vector has a versatile polylinker that allows in-frame cloning of the receptor ECD with the mouse IgG1 sequence to encode a receptor ECD-IgG1 fusion construct. The receptor-IgG1 fusion proteins are transiently expressed in useful amounts following transfection of the expression vector into COS7 cells and G418 selection. The mouse IgG1 portion of the fusion protein provides a universal handle for purification on an affinity matrix and detection by anti-mouse IgG antibodies in ELISA or Western blot formats. The expressed receptor ECD-IgG1 fusion proteins bind their cognate ligands. In order to demonstrate that the fusion proteins have similar ligand binding affinities as the native receptors, the affinity constants (Kd) for EPOR, TNFR, IL-4R, and IL-6R-IgG1 fusion proteins were measured by surface plasmon resonance and shown to be in good agreement with published values. The TNFR-IgG1 fusion protein was employed in a demonstration of a novel ELISA format for detecting cytokine receptor binding to cytokine.

Increased protein expression through improved ribosome-binding sites obtained by library mutagenesis.

This report describes a method whereby library mutagenesis combined with drug selection was used to generate unique and efficient ribosome-binding sites (RBS) for expressing recombinant proteins in Escherichia coli. The RBS was deleted from a vector expressing beta-lactamase and replaced with a 16-base sequence containing a library of mutations. Selection of the library with ampicillin yielded several unique RBS sequences that were more efficient than ompA RBS for expressing a bacterial (beta-lactamase) and a mammalian protein (single-chain Fv antibody). The described approach provides a practical means to improve recombinant protein expression and, also, provides new sequences to further evaluate the complex regulatory mechanism underlying translation initiation.

Increased antibody expression from Escherichia coli through wobble-base library mutagenesis by enzymatic inverse PCR.

We tested the value of a new library mutagenesis approach, called library enzymatic inverse PCR (LEIPCR), for expression-level enhancement of antibody Fv fragments produced in Escherichia coli. The production level of active, metal chelate-specific antibody from our constructs is limited by a low expression level of the second, heavy-chain cistron. To increase the production level, LEIPCR was applied to the wobble bases of the second cistron leader peptide. In LEIPCR mutagenesis, the entire plasmid is amplified using mutagenic primers with class-IIS restriction endonuclease (ENase) sites at their 5' ends. The PCR product is digested with the class-IIS ENase (here, BsaI; GGTCTCN[symbol: see text]NNNN[symbol: see text]), which removes its own recognition sequence, and the ends are self-ligated. Thus, LEIPCR can be used to make plasmid mutant libraries regardless of the nucleotide sequence, and independent of available ENase sites. The resulting library of 10(7) wobble mutants was screened for active Fv by a colony filter lift. A selected mutant was shown to produce between four- and elevenfold more active Fv than the wild type (wt), and fivefold more heavy chain. Mutations outside of the leader peptide were shown not to be involved. The mutated areas of the mRNAs of two different up-mutants may have less secondary structure than the wt. Thus, the sequence of the mRNA of the second leader peptide was limiting to the expression level of heavy-chain and active Fv.