Enhanced protease cleavage efficiency on the glucagon-fused interleukin-2 by the addition of synthetic oligopeptides.
Human interleukin-2 (hIL-2) was produced as a recombinant fusion protein (G3.IL-2/HF) consisting of three tandem-arranged human glucagon molecules (G3) and hIL-2. For the recovery of hIL-2, a factor Xa (FXa) cleavage sequence was introduced next to the N-terminus of hIL-2. Cleavage efficiency on this recombinant protein construct was very low because its recognition sequence was sterically hindered within the G3.IL-2/HF molecule and hence FXa access to the cleavage site was insufficient. We therefore introduced various synthetic oligopeptides upstream from the FXa cleavage site as a means to change substrate conformation and thereby increase cleavage efficiency. Among these oligopeptides, acidic or nucleophilic constructs were the most effective for the FXa-mediated cleavage of the fusion protein. In addition, insertion of various oligopeptides into the G3.IL-2/HF molecule varied the solubility of each construct depending on their physical properties. Consequently, the G3.IL-2/DF construct showed the highest final hIL-2 yields via FXa-mediated removal of the fusion partner. Lastly, we confirmed that cleavage efficiency was greatly increased but native hIL-2 was cleaved internally by non-specific cleavage when the acidic oligopeptide D4 (DDDD) was introduced upstream of the EK cleavage site within G3.IL-2/HE molecule. The G3.IL-2/HE molecule was shown to be an inefficient substrate to EK in a previous report (Biotechnol. Bioprocess Eng. (2000) 5, 13-16).
Kim
Sung-Woo
SW
AceBiotech Co., Ltd., #114 Bio-Venture Center (BVC), KRIBB, Yuseong, Daejeon 305-806, Republic of Korea.
Kim
Jae-Bum
JB
Lee
Weon Sup
WS
Jung
Woo-Hyuk
WH
Ryu
Ji-Myung
JM
Jang
Hyung-Wook
HW
Jo
Young-Bae
YB
Jung
Joon-Ki
JK
Kim
Jung-Hoe
JH
D003546:Cysteine Endopeptidases / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005934:Glucagon / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility
D007376:Interleukin-2; D009363:Neoplasm Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D005934:Glucagon; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant