David John Chiswell

Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display

Functional antibody fragments may be displayed on the surface of filamentous bacteriophage by introducing variable region genes into the viral genome at a gene encoding a viral coat protein. “Phage display” enables the isolation of antibody genes from large libraries according to the binding specificities they encode. We have constructed a new phage-display vector encoding a polyhistidine tag that has been used for rapid purification of soluble antibody fragments. An antibody library derived from immunized mice was cloned into this vector. This library was panned against the transition state analog RT3, and a high proportion of binders isolated after two rounds of panning. PCR analysis revealed that there were 24 different pattern groups. Sequencing of 15 clones within the major pattern group revealed 10 related clones with a range of point mutations. Thus, phage display can provide a large diverse repertoire of candidate catalytic antibodies based on TSA selection and screening.

Molecular mechanisms involved in morphological variation of avian sarcoma virus-infected rat cells.

Clones of avian sarcoma virus (ASV)-infected rat cells are heterogeneous, containing both morphologically transformed and morphologically normal cells. Morphological variation is reversible: normal cells can give rise to transformed daughters and they in turn can segregate morphologically normal revertants. However, reversion of transformed clones seems to occur more readily than transformation of normal cells. In most cases, morphologically normal cells contain recoverable viruses with no defect in transformation and yet the cells themselves are usually fully susceptible to transformation by superinfecting ASV. With one exception, morphological changes are not accompanied by detectable alterations in the size or chromosomal location of the single ASV provirus that these clones possess. In one clone, All, that has been examined in detail, transformed segregants contain virus specific RNAs and the viral transforming protein, pp60src, whereas in normal subclones neither RNA nor protein can be detected. These results indicate that morphological variation reflects a reversible modulation in expression of viral functions, probably specific to individual proviruses and probably operating at the level of viral RNA transcription.

Human antibody engineering: Current Opinion in Structural Biology 1993, 3:564–571

Abstract The most recent developments in manipulation and selection of antibody genes indicate that the next generation of antibody-based products, particularly those destined for human therapy, could be created entirely in vitro.

Mapping of nonconditional and conditional mutants in the src gene of Prague strain Rous sarcoma virus

Abstract Restriction endonuclease Eco RI digestion of the viral DNA of 12 nonconditional transformation defective ( td ) mutants of Prague strain Rous sarcoma virus (PR-RSV) has divided these mutants into two groups. Five mutants possess an Eco RI B ( src gene-containing) fragment of the same size as that from wild type PR-RSV and thus these mutants have no detectable diminution in the transforming src gene. The other 7 mutants bear deletions of 1.0 to 1.8 kilobases in the 3.2-kilobase Eco RI B fragment. The extents of these deletions have been mapped using a number of restriction endonucleases and by comparing these results with studies on the nucleotide sequence of src (Czernilovsky et al., Nature (London) 287 , 198–203, 1980) we conclude that the td mutants have deleted sequences at the 5′ end of src , and in some cases also in regions between src and env , leaving intact at least some 3′ src sequences. These td mutants recombine in differing patterns with 14 temperature-sensitive ( ts ) src gene mutants. This enables many of the ts mutations to be localized in limited regions of src , 10 of them being clustered in the 3′ 40% of the gene, the remaining four bearing at least one mutation in the 5′ 60% of src . A nonconditional src gene mutant that transforms cells to a fusiform as opposed to round cell morphology ( td SF/LO 104) also possesses a lesion that maps in the 5′ 60% of the src gene.