Construction of Escherichia coli vectors for expression and mutagenesis: synthesis of human c-Myc protein that is initiated at a non-AUG codon in exon 1.
Three types of Escherichia coli vector for both gene expression and mutagenesis were constructed from a plasmid/phage chimera vector pUC118. Each vector contains the lac (pTD-lac), tac (pTD-tac), or T7 promoter (pTD-T7). Downstream from the promoter, these vectors have sequences in common, including a Shine-Dalgarno (SD), multiple cloning sequence, sequence-primer binding site, transcription termination signal, and M13 origin of replication. Using single-stranded circular DNA obtained by infection with helper phage, oligodeoxyribonucleotide (oligo)-directed mutagenesis allows the appropriate fusion between the vector SD sequence and the start codon in the inserted fragment. Since a complementary oligo representing a large deletion is generally used for this construction, the extra nucleotides in the opposing strand form a loop structure. Thus, we have designated this mutagenesis as 'loop-out mutagenesis'. Expression plasmid encoding the larger human c-Myc protein that is initiated at a non-AUG codon in exon 1 and its derivatives were constructed using a pTD-T7 vector. Expression experiments indicated that the wild-type (wt) protein was synthesized poorly after induction with isopropyl-beta-D-thiogalactopyranoside, while one of the derivatives, p62M1T, in which a threonine residue was added at the N terminus of the wt protein, was produced in a large quantity in E. coli.
Medical Research Institute, Kanazawa Medical University, Ishikawa, Japan.
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D001483:Base Sequence; D003062:Codon; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010442:Peptide Chain Initiation, Translational; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc
D003062:Codon; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc