High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling.
Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.
Tenno
Takeshi
T
Graduate School of Integrated Science, Yokohama City University, 1-7-29 Suehirocho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan.
Goda
Natsuko
N
Tateishi
Yukihiro
Y
Tochio
Hidehito
H
Mishima
Masaki
M
Hayashi
Hidenori
H
Shirakawa
Masahiro
M
Hiroaki
Hidekazu
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D015951:Factor Xa / Q000378:metabolism; D005822:Genetic Vectors; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D010455:Peptides; D004247:DNA; D015951:Factor Xa