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During the last decade, recombinant antibody engineering has emerged as one of the most promising approaches for the design, selection and production of molecules for basic research, medicine and the pharmaceutical industry. This MiniReview describes the major findings that have led to the development of this powerful technique, with an emphasis on the use of Escherichia coli and filamentous phage as a tool allowing powerful selection procedures from large libraries as well as the use of intracellular expression of antibody fragments as a new class of neutralizing molecules with a potential use in therapy. The future of these rapidly evolving technologies is discussed.

International audience; Mutants of colicin A have been prepared in which the three tryptophan residues (Trp86, Trp130 and Trp140), localized in the C-terminal domain of the soluble wild-type protein, have been substituted by phenylalanine. The Trp140Phe single mutation had the effect of decreasing the percentage of protein that is expressed as insoluble aggregates. The created hydrophobic cavity decreased the stability of the protein during its folding, resulting in partial aggregation in the cytoplasm of the Escherichia coli-producing cells. Aggregation was increased when Trp140 was substituted by Lys, Leu or Cys, or if the Trp140 mutation was combined with the Trp86Phe and/or Trp130Phe mutations. A single mutation, Lys113Phe, however, was able to restore the solubility of the aggregated mutants in vivo. Detailed analysis of the 3-D structure of the C-terminal domain of colicin A suggests that filling of the hydrophobic cavity is responsible for this effect.

We have constructed deletion and point mutations within the Simian virus 40 (SV40) early promoter region which contains two tandemly repeated 21 bp sequences and a related 22 bp sequence (the "upstream" 21 bp repeat region). After transfection into permissive CV-1 cells and non-permissive mouse 3T3-4E cells, the effect of the mutations on early gene expression was studied by measuring T-antigen production, using indirect immunofluoresence. Our results demonstrate that the 21 bp repeat region, and in particular the six GC-rich motifs 5'-CCGCCC-3' which are repeated in this region constitute an important element of the SV40 early promoter. Surprisingly, we found that the requirement for the 21 bp repeat region for early gene expression was partially fulfilled even when it was in the inverted orientation.

In Escherichia coli cells, cytoplasmic proteins are produced in free polysomes whereas exported proteins, like periplasmic and outer membrane proteins are produced in membrane-bound polysomes [1-3]. As a consequence, synthesis of exported proteins is preferentially inhibited by low concentrations of spectinomycin and tetracycline [4]. It has been demonstrated that the interaction of ribosomes and membrane in membrane-bound polysomes is mainly, if not only, mediated by nascent polypeptide chains [3]. Lipoprotein, first isolated by Braun and Rehn [5] is the most abundant protein of the outer membrane because about 7.5.105 copies can be found, partly bound to the murein and partly free [6]. This protein is produced in a precursor form and very likely in the membrane-bound polysomes since it has been demonstrated that the ratelimiting step for the assembly is the rate of synthesis [7]. Two out of three fatty add chains are bound to the protein in a precursor form, the third binds to the amino terminal residue, formed after cleavage of the signal peptide [8]. Due to the short size of prolipoprotein (78 amino acids) one can calculate that only 2 to 4 ribosomes at any given time are bound to the mRNA [9]. Furthermore, about 40 amino acid residues are masked in the large subunit of ribosomes [10]. Therefore, the question arises to know in what polysome fraction membrane-bound) this protein is produced in vitro after separation of these polysomes. In this study we have addressed this question and evidence has been obtained that the lipoprotein is produced in cell-free systems programmed by free-polysome fraction. The significance of this' result is discussed.