Simple purification of a foreign protein using polyhedrin fusion in a baculovirus expression system.
Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures, and that these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with the Polh gene at the N-terminus, including a linker and enterokinase (EK) site between Polh and EGFP, was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, EGFP presented mainly in the supernatant, and this supernatant fraction also showed a pure EGFP band. These results suggest that a combined procedure of Polh fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.
Roh
Jong Yul
JY
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea.
Choi
Jae Young
JY
Kang
Joong Nam
JN
Wang
Yong
Y
Shim
Hee Jin
HJ
Liu
Qin
Q
Tao
Xueying
X
Xu
Hong Guang
HG
Hyun
Jin-Ho
JH
Woo
Soo Dong
SD
Jin
Byung Rae
BR
Je
Yeon Ho
YH
D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins