Thomas Dr Seehaus

A surface expression vector for antibody screening.

To select specific antibodies (Ab) from large recombinant libraries using small amounts of antigen, we have constructed a phagemid that expresses a single-chain Ab fused to pIII, a coliphage protein product of gene III that initiates infection by binding to F pili. Surprisingly, the production of the fusion protein (Ab::pIII) was induced by wild-type (wt) phage fd in the absence of IPTG. Ab::pIII was identified by a monoclonal Ab to an epitope in the linker sequence between the heavy and light chains, and by antisera to their N-terminal sequences. It is able to bind antigen and be assembled into infectious phagemid particles that can be enriched on columns of immobilised antigen. The phagemid DNA is even smaller than that of wt fd phages and can easily be propagated in plasmid form. Most importantly, its Ab::pIII-encoding gene can be tightly repressed so that Ab libraries can be amplified without risk of being dominated by deletion mutants. After induction, however, large quantities of the fusion protein can be produced, thus greatly facilitating its analysis.

Comparative analysis of tubulin sequences

1. Information on the structure and evolution of tubulin has been obtained by comparing the available sequence data on 31 alpha-tubulins and 31 beta-tubulins. 2. Similar numbers of conserved amino acids are found amongst both alpha- and beta-tubulins (alpha: 48%, plus conservative substitutions: 72%; beta: 48%, plus conservative substitutions: 70%). About half of them are common to both subunits (23%, plus conservative substitutions: 45%). Four cysteines in the alpha-tubulins and 2 cysteines in the beta-tubulins are conserved. Only one cysteine (position 129) is conserved in all alpha- and beta-tubulins. 3. The longest unbroken stretch of identical amino acids between all the alpha- and beta-tubulins is found in positions 180-186 (Val-Val-Glu-Pro-Tyr-Asn), a region that appears to be important for binding the ribose moiety of GTP. Two other groups of amino acids implicated in GTP binding, one near position 70 and a glycine cluster at position 144 are also quite conserved. 4. Extra length differences between tubulin subunits, presumably present as extensions on the dimer surface, have been observed at position 50 and near position 360 in alpha-tubulins and in one case at position 57 in a beta-tubulin. 5. The introns of tubulin genes, many of them clustered in the first quarter of the tubulin coding region, do not appear to correspond to any particular structural or functional regions. 6. Mutation rates of tubulins vary considerably. The lowest alpha-tubulin homology (62.3%) is between a very divergent Drosophila alpha-tubulin and an alpha-tubulin from the yeast S. cerevisiae. The lowest beta-tubulin homology (63.3%) is between a yeast (S. cerevisiae) beta-tubulin and a mouse beta-tubulin expressed in hematopoietic tissue. In contrast, some mammalian and bird tubulins are almost identical. 7. Tubulins heterogeneous C-termini are useful for identifying corresponding tubulins of different vertebrate species, many of which are remarkably conserved. Exceptions are the divergent beta-tubulins of erythrocyte and thrombocyte marginal bands. 8. We have proposed a model for tubulin evolution in metazoan organisms in which the release of structural constraints after gene duplication is a major cause of relatively rapid change.