The N-terminal portion (aa 4-77) of CYB-1 was cloned into a maltose-binding protein (MBP)-tagged 6xHis vector, pYS, as follows. First, the MBP gene was PCR-amplified from pHIT198 (a gift from H. Tabara, Tsukuba University, Kyoto, Japan) and cloned into pET- 28a(+) (Novagen, 69864-3) by means of the DNA Ligation Kit Mighty Mix (Takara, 6023) to generate pYS. Second, the N-terminal portion (aa 4-77) of CYB-1 was amplified from yk1306b09 (a gift from Y. Kohara, National Institute of Genetics, Mishima, Japan) and cloned into the pYS vector. The CYB-1-MBP-6xHis fusion protein was expressed and affinity-purified using His-Bind Agarose Resin (ELPIS-BIOTECH, EBE-1031). It was then injected into rats as an antigen to raise an anti-CYB-1 antibody (AbFrontier). This antibody was then affinity-purified from the crude serum, using 6xHis-tagged CYB-1-GST. The GST gene was amplified from pDEST15 (Invitrogen, 11802-014) and inserted into pET-28a(+) (Novagen, 69864-3) to generate pYS1. The same N-terminal portion of CYB-1 used to produce the CYB-1-MBP-6xHis fusion protein was cloned into the pYS1 vector.
cyb-1