Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli

• Published in: Gene Vol. 69; no. 2; pp. 301 - 315
• Main Authors: Amann, Egon, Ochs, Birgit, Abel, Karl-Josef
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 30.09.1988; Amsterdam Elsevier; New York, NY
• Subjects: anticoagulant protein PP4; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Base Sequence; Biological and medical sciences; Biotechnology; cat gene; Codon; Escherichia coli; Escherichia coli - genetics; eukaryotic protein overproduction; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genes; Genes, Bacterial; Genetic engineering; Genetic technics; Genetic Vectors; Genotype; human coagulation factor XIIIa; lac operator; lacI-coded repressor; Methods. Procedures. Technologies; Molecular Sequence Data; Mutation; Plasmids; Promoter Regions, Genetic; Recombinant DNA; Recombinant Fusion Proteins - biosynthesis; Recombinant Proteins - biosynthesis; Restriction Mapping; trc promoter; Vectors (cloning, transfer, expression). Insertion sequences and transposons; lacI-coded repressor; dNTP; Cm; Recombinant DNA; lac operator; bp; Ap; Tc; eukaryotic protein overproduction; R; S; anticoagulant protein PP4; human coagulation factor XIIIa; kb; oligo; Tn; aa; RBS; SDS; cat gene; PAGE; plasmids; PolIk; LB; CAT; IPTG; trc promoter; Human; Regulation(control); Transcription promoter; Escherichia coli; Factor XIII; Bacteria; Anticoagulant; Fusion protein; Gene expression; Escherichieae; Vector; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(88)90440-4

A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183–190], the vectors carry a strong hybrid trp/lac promoter, the lacZribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacI q allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.