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 purifi...

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Published in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2010, Vol.74 (8), p.1522-1526
Main Authors: ROH, Jong Yul, CHOI, Jae Young, KANG, Joong Nam, WANG, Yong, SHIM, Hee Jin, LIU, Qin, TAO, Xueying, XU, Hong Guang, HYUN, Jin-Ho, WOO, Soo Dong, JIN, Byung Rae, JE, Yeon Ho
Format: Article
Language:English
Subjects:
Baculoviridae - genetics
baculovirus expression system
Base Sequence
Biological and medical sciences
Cell Line
easy purification system
enhanced GFP (EGFP)
Fundamental and applied biological sciences. Psychology
Gene Expression
granulation
Green Fluorescent Proteins - genetics
polyhedrin fusion
Protein Engineering - methods
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
Viral Structural Proteins - biosynthesis
Viral Structural Proteins - chemistry
Viral Structural Proteins - genetics
Viral Structural Proteins - isolation & purification
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Summary: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.
ISSN:0916-8451
1347-6947
DOI:10.1271/bbb.100016