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Biomedical subjects

E Remaut

Publications and source records attributed to E Remaut.

At least 37 records · Page 2Linked to original sources
PubMed1993

LamB as a carrier molecule for the functional exposition of IgG-binding domains of the Staphylococcus aureus protein A at the surface of Escherichia coli K12.

One, two or four IgG-binding domains of the Staphylococcus aureus Protein A (SPA) were inserted into the LamB protein which was expressed under control of the tac promoter. The chimeric proteins were shown to be exposed at the cell surface by analysis of isolated outer membranes and also by testing their functional interaction with IgG molecules. We hereby show that the LamB protein can accept as many as 232 amino acids (four SPA domains) and still be incorporated into the Escherichia coli outer membrane, while maintaining the functional conformation of the inserted SPA polypeptides.

Bacterial Outer Membrane Proteins↗
PubMed1993

Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of Staphylococcus aureus in Escherichia coli.

Fusion genes between papA, the gene coding for the major Pap pilus subunit, and fragments coding for an immunoglobulin G-binding domain of the Staphylococcus aureus protein A were constructed in such a way that the spa fragments were inserted following either codon 7 or 68 of the coding sequence for the mature portion of PapA. Peptides in the area of amino acids 7 and 68 of PapA are localized at the external side of the pilus. A set of pL expression plasmids containing papA and derivatives suitable for insertion were constructed. A papA gene carrying a spa insert following codon 68 was cloned back into the pap operon. The presence of this altered operon in a bacterial strain allowed the detection of immunoglobulin G-binding activity at the surfaces of the bacterial cells.

Amino Acid Sequence↗
PubMed1992

Disulphide bridge formation in the periplasm of Escherichia coli: beta-lactamase:: human IgG3 hinge fusions as a model system.

We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.

Bacterial Proteins↗
PubMed1989

Correlation between temperature-dependent cytoplasmic solubility and periplasmic export of a heterologous protein in Escherichia coli.

The coding sequence of mature human tumor necrosis factor (hTNF) was fused to the signal-encoding sequence of beta-lactamase (Bla). Mature hTNF was exported into the periplasm of Escherichia coli. A mutant hTNF [Van Ostade et al., FEBS Lett. 238 (1988) 347-352], which displays a temperature-dependent intracellular solubility, was fused to the same Bla signal-encoding sequence. We found that the export competence of the mutated hTNF was correlated with the intracellular solubility of this protein. We postulate that the secretion proficiency of eukaryotic proteins, when fused to a prokaryotic export signal, depends on the ability of the mature protein to readily fold into a soluble conformation.

Amino Acid Sequence↗
PubMed1987

A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species.

We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon. The vector carries the strong leftward promoter (pL) of coliphage lambda as well as the cI857 allele, which codes for a thermolabile repressor protein. The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter. The plasmid was introduced into Erwinia and Serratia species by means of mobilization. Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera. As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form.

Bacteriophage lambda↗
PubMed1986

Inefficient translation initiation causes premature transcription termination in the lacZ gene.

Expression plasmids containing the E. coli lacZ coding region preceded by a set of different ribosome-binding sites and put under transcriptional control of the leftward promoter of phage lambda (PL) were used to study the synthesis of lacZ mRNA. In a normal host the steady state level of full-length lacZ mRNA varied 100-fold with the different synthesis levels of beta-galactosidase, whereas in a host expressing the antitermination protein N of phage lambda, all vectors synthesized the same amount of full-length lacZ mRNA, while maintaining the differences in beta-galactosidase expression. We present evidence for a causal relationship between the rate of ribosome loading and the continuation of transcription across the lacZ gene. We suggest that extended spacing between the RNA polymerase and the elongating ribosome causes transcriptional polarity by increasing the extent of premature termination. The conditional character of the termination event can best be explained by invoking termination factor Rho.

Galactosidases↗
PubMed1986

Cloning and structure of a mouse interleukin-2 chromosomal gene.

Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, +/- 2 400, and +/- 1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin-2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consecutive CAG codons coding for glutamine, is found in the first exon.

Amino Acid Sequence↗
PubMed1986

[Preparation and characterization of antisera specific to various polypeptide domains corresponding to the v-ets oncogene of the avian leukemia virus E26].

We prepared antisera to three distinct portions of the v-ets oncogene of the avian leukemia virus E26. An antiserum directed against the middle v-ets-encoded domain identifies in different chicken cell lines and normal tissues a c-ets-encoded protein of Mr 54,000 (P54c-ets) and three proteins of Mr 60,000 62,000 and 64,000 partially related to P54c-ets. Antisera directed against the aminoterminal v-ets-encoded domain failed to precipitate P54c-ets or P60/P64. Thus, the E26 specific v-ets oncogene displays a complex structure that includes several distinct portions, the genetic origin of which could be different.

Animals↗
PubMed1985

Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression.

A vector containing the leftward promoter (pL) as transcription initiation signal and a synthetic, easily adaptable translation initiation region have been constructed. We have used the expression system to assess the relevance of sequences upstream from the Shine-Dalgarno (SD) region in the translational-initiation process. To this end, a series of structural variants of the prototype ribosome-binding site were used to direct the synthesis of both mature human fibroblast interferon and beta-galactosidase (beta-gal). It was found that alterations 5' to the SD element can considerably affect the rate of mRNA translation. The observation that the relative efficiency of the various 5'-untranslated regions depends on the downstream coding information implies that secondary (and/or tertiary) structure formation is of major importance in the initiation process. But an mRNA folding, in which the SD and ATG determinant are set free in single-stranded regions, does not unconditionally guarantee an efficient initiation of translation.

Base Sequence↗
PubMed1984

Oligonucleotide directed mutagenesis: selection of mutants by hemimethylation of GATC-sequences.

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an AT----CG transversion in the mut L region of phage lambda. Under optimal conditions, about 50-60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.

Bacteriophage lambda↗
PubMed1984

High-level expression of human interferon gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda.

Several recombinant plasmids have been constructed which direct high-level synthesis of mature human interferon gamma (IFN-gamma) in Escherichia coli using the inducible leftward promoter pL of phage lambda followed by a translational initiator region derived either from the phage MS2 replicase gene or the E. coli tryptophan attenuator region. Under these conditions, IFN levels of up to 25% of the total cellular protein can be achieved. The highest levels were obtained when a terminator of transcription was cloned downstream from the IFN-gamma sequence. IFN-gamma was almost entirely found in the initial pellet fraction and not in soluble extracts. Co-induction of the lysis genes derived from phage MS2 or from phage lambda, inserted downstream from the IFN-gamma sequence, did not enhance the biological activity present in the supernatant fraction.

Bacteriophage lambda↗
PubMed1983

Codon usage and mistranslation. In vivo basal level misreading of the MS2 coat protein message.

The coat protein of the small RNA virus MS2 shows charge heterogeneity in vivo. In most strains there is a basic satellite of the native protein. We have shown that this basic satellite is greatly diminished or absent in strains with the streptomycin-resistant allele, rpsL, a mutation which leads to increased translational accuracy. Further, the satellite is present in cells where the coat protein is encoded by duplex DNA. Tryptic digests of the satellite show that it contains new lysine-containing peptides which appear to be the same as those found in derivatives of coat protein which have a lysine for asparagine substitution. Sequencing of the NH2-terminal 19 amino acids of the satellite protein shows that the asparagine codon AAU at amino acid 12 is misread approximately 8 times more frequently than the AAC at amino acid 3. We conclude that the satellite species is the result of basal level lysine for asparagine substitution. These substitutions are most likely caused by preferential misreading of AAU codons at a frequency of approximately 5 X 10(-3), 10-fold higher than the average error frequency.

Amino Acid Sequence↗
PubMed1983

Inducible high level synthesis of mature human fibroblast interferon in Escherichia coli.

We have obtained high level synthesis in Escherichia coli of mature human fibroblast interferon using a plasmid vector that was designed to allow easy coupling of a DNA coding region to the initiator AUG of the replicase gene of the RNA phage MS2 cloned downstream of phage lambda's leftward promoter. The activity of the promoter can be regulated by temperature. Induced cells accumulated the interferon up to 4% of the total cellular protein. The biological activity of the product amounted to 4 X 10(9) international units per litre of culture. The synthesis of human fibroblast interferon was shown to drastically inhibit the growth rate of the bacterial host.

Bacteriophage lambda↗
PubMed1983

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli.

A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.

Amino Acid Sequence↗
PubMed1983

Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication.

Improved expression vectors have been constructed which are derived from runaway-replication mutants of plasmid R1 and carry the strong leftward promoter (pL) of bacteriophage lambda. The activity of this promoter is controlled by a temperature-sensitive repressor, product of the phage gene cI cloned on a compatible plasmid. Heat induction leads to amplification of the plasmid copy number and at the same time turns on the promoter. At a short distance downstream from the promoter, unique EcoRI, BamHI, XbaI and HindIII sites are present. This system was used for high level expression of the T4 DNA-ligase gene; 3 h after induction the ligase amounted to about 20% of total cellular protein.

Bacteriophage lambda↗