Erik Remaut

Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda

Abstract A family of plasmid cloning vectors has been constructed that make use of the leftward promoter ( P L ) of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of P L is fully repressed at low temperature by a thermolabile repressor product of the λc I 1857 gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under P L control.

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.

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.

Inefficient translation initiation causes premature transcription termination in the IacZ 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.

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.

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.

SummaryOne, 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.

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 consective CAG codons coding for glutamine, is found in the first exon.

Studies on the bacteriophage MS2. XVI. The termination signal of the A protein cistron.

The RNA of bacteriophage MS2 codes for three viral proteins: the coat protein, the A protein and the replicase. Upon infection of various amber suppressor strains of Escherichia coli, we found a fourth viral protein, the synthesis of which was specifically dependent on the presence of an amber suppressor gene. It is shown that this polypeptide is formed by reading through the natural termination signal of the A protein cistron. This cistron therefore terminates with the nonsense codon UAG. The observed prolongation accounts for the addition of some 30 amino acids. Unlike the normal A protein, the longer polypeptide is probably not incorporated into mature phage particles. In the related phage Qβ, no similar prolongation of the A protein was observed.

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

SummaryWe 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 nut L region of phage λ. 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.

Studies on the bacteriophage MS 2: XVII. Suppressor-sensitive mutants of the a protein cistron

SummaryThe viral proteins synthesized in non-suppressor cells by amber mutants in the A protein cistron of the RNA bacteriophage MS2 were analyzed. Protein synthesis was studied in rifampicin-inhibited cultures and the labeled, viral proteins were separated on sodium dodecyl sulphate containing polyacrylamide gels. We found that 7 out of 19 mutants synthesized an A protein-fragment corresponding in length to 88% of the wild-type A protein. This fragment was not incorporated into the defective particles formed by the mutants. 12 mutants synthesized no detectable amount of fragment. It was shown that the absence of fragment is not due to selective proteolytic breakdown.