Biomedical subjects

E Ferrando

Publications and source records attributed to E Ferrando.

3 recordsLinked to original sources

PubMed1993

Homologous bacterio-opsin-encoding gene expression via site-specific vector integration.

Homologous recombination in the archaebacterium Halobacterium halobium has been investigated and exploited for the wild-type (wt) level of expression of the bacterio-opsin-encoding gene (bop). The Haloferax volcanii-Escherichia coli shuttle vector, pWL102, was used to construct a shuttle-mutagenesis vector, pEF191, bearing bop and short flanking sequences. Transformation of a bacteriorhodopsin (BR)-negative H. halobium strain with pEF191 resulted in plasmid integration at the homologous bop locus. A model for this site-specific vector integration is presented which has been confirmed by determining the arrangement of the repeated homologous sequences on the chromosome. Two different configurations are obtained after integrative transformation due to the presence of an insertion element in the genomic copy of bop. In one configuration, the functional bop cluster containing the regulatory bat and brp genes was in wt arrangement. In the second configuration, the bop cluster is interrupted by 10 kb of plasmid vector sequences, and the upstream region required for bop expression was limited to 400 bp. The BR production for both configurations was determined and found to be at wt level. These results suggest that the function of the putative bop promoter does not depend on the defined upstream positions of bat and brp. The system presented here can be easily exploited for structure-function studies on BR and introduces homologous gene targeting as a powerful tool in the study of halobacterial genetics.

Bacteriorhodopsins↗

PubMed1992

General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase.

A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.

Amino Acid Sequence↗

PubMed1989

Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor.

The gene coding for sensory rhodopsin I (SR-I) has been identified in a restriction fragment of genomic DNA from the Halobacterium halobium strain L33. Of the 1014 nucleotides whose sequence was determined, 720 belong to the structural gene of SR-I. In the 5' non-coding region two putative promoter elements and a ribosomal binding site have been identified. The 3' flanking region bears a potential terminator structure. The SR-I protein moiety carries no signal peptide and is not processed at its N terminus. The C terminus, however, lacks the last aspartic acid residue encoded by the gene. Analysis of the primary structure of SR-I reveals no consistent homology with the eukaryotic photoreceptor rhodopsin, but 14% homology with the halobacterial ion pumps, bacteriorhodopsin (BR) and halorhodopsin (HR). Residues conserved in all three proteins are discussed with respect to their contribution to secondary structure, retinal binding and ion translocation. The aspartic acid residue which mediates in BR the reprotonation of the Schiff base (D96) is replaced in SR-I by a tyrosine (Y87). This amino acid replacement is proposed to be of crucial importance in the evolution of the slow-cycling photosensing pigment SR-I.

Amino Acid Sequence↗