title: Simple purification of a foreign protein using polyhedrin fusion in a baculovirus expression system.
abstract: 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.
authors.affiliation: Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea.
authors.forename: Jong Yul
authors.initials: JY
authors.lastname: Roh
authors.forename: Jae Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Joong Nam
authors.initials: JN
authors.lastname: Kang
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Hee Jin
authors.initials: HJ
authors.lastname: Shim
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.forename: Xueying
authors.initials: X
authors.lastname: Tao
authors.forename: Hong Guang
authors.initials: HG
authors.lastname: Xu
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Hyun
authors.forename: Soo Dong
authors.initials: SD
authors.lastname: Woo
authors.forename: Byung Rae
authors.initials: BR
authors.lastname: Jin
authors.forename: Yeon Ho
authors.initials: YH
authors.lastname: Je
mesh_terms: 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*
chemical_list: D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins