Expression of a synthetic gene encoding human transthyretin in Escherichia coli.
Transthyretin is an amyloidogenic protein that causes human amyloid polyneuropathy and senile systemic amyloidosis as a result of the deposition of normal and/or mutant transthyretin in the form of amyloid fibrils. A high-expression plasmid of human transthyretin was constructed in order to facilitate the study of amyloid fibril formation of this protein. The transthyretin gene was constructed by an assembly of eight chemically synthesized oligonucleotides and amplified by polymerase chain reaction, and the amplified gene was inserted into an Escherichia coli expression vector. The expression plasmid was transformed into M15 cells and the gene product was expressed as a polyhistidine-tagged fusion protein. Purified recombinant transthyretin was obtained by one-step nickel chelation affinity chromatography and the production level of the protein was 130mg per 1L of culture. Furthermore, the expressed protein showed the same characteristics in terms of tetramer formation at neutral pH and amyloid formation at acidic pH as did the authentic human transthyretin. This system will enable biophysical and structural studies of this protein to be advanced.
Matsubara
Kimiaki
K
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Mizuguchi
Mineyuki
M
Kawano
Keiichi
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic / Q000235:genetics*; D005818:Genetic Engineering; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011228:Prealbumin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D011228:Prealbumin; D011994:Recombinant Proteins; D006639:Histidine