Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle.
A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.
Takasuga
A
A
Tsukuba Research Laboratory, Hitachi Chemical Co., Ltd., Ibaraki.
Banba
K
K
Yoshino
K
K
Izutsu
H
H
Iwakura
M
M
Ohashi
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007073:Immunoglobulin E / Q000096:biosynthesis / Q000235:genetics*; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D014357:Trypsin
D000596:Amino Acids; D007141:Immunoglobulin Fc Fragments; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C079100:aspartyl-seryl-aspartyl-glycyl-lysine; D007073:Immunoglobulin E; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D014357:Trypsin