Improvement of GFPuv-beta 3GnT2 fusion protein production by suppressing protease in baculovirus expression system.
The effects of protease inhibitors on the production of recombinant protein were investigated using a recombinant baculovirus containing GFPuv-human beta 1,3-N-acetylglucosaminyltransferase 2 (beta 3GnT2) connected to the prepromelittin signal sequence. The addition of leupeptin as a cysteine protease inhibitor at 2.5 microg/ml improved intra- and extracellular beta 3GnT activities 5- and 3-fold, respectively, compared to those without addition, which was due to a suppression of protease activity. With the leupeptin addition only four degraded molecular bands lower than 32 kDa appeared, but 9 degraded molecular bands between 29 kDa and 41 kDa existed without addition. In contrast, pepstatin A as a carboxyl protease inhibitor had no influence on the improvement of beta 3GnT production, judging from SDS-PAGE. Moreover, when 50 microM carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132), known as a proteasome inhibitor, was used in combination with the leupeptin, a ladder of low molecular mass bands of fusion protein was diminished. The intracellular beta 3GnT activity increased 9-fold, to as high as that without addition of two kinds of protease, but the extracellular activity was not different from that with the addition of only leupeptin. These findings indicate that the decrease in cell viability causes the decrease in the secretion rate of intracellular fusion protein, resulting the accumulation of the full-length of fusion protein.
Kato
Tatsuya
T
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Japan.
Murata
Takeomi
T
Usui
Taichi
T
Park
Enoch Y
EY
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D014466:Ultraviolet Rays
D017931:DNA Primers; D008164:Luminescent Proteins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017351:N-Acetylglucosaminyltransferases