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Research article

J. Microbiol. Biotechnol. 2010; 20(3): 460-466

Published online March 28, 2010 https://doi.org/10.4014/jmb.0908.08013

Copyright © The Korean Society for Microbiology and Biotechnology.

Protein Cyclization Enhanced Thermostability and Exopeptidase-Resistance of Green Fluorescent Protein

Zhonglin Zhao 1, Xin Ma 1, Liang Li 1, Wei Zhang 1, Shuzhen Ping 1, Ming-Qun Xu 2 and Min Lin 1*

1 Biotechnology Research Institutee, Chinese Academy of Agricultural Sciencess, Beijing 100081, P. R. China , 2New England Biolabss, Inc., Ipswich, MA 01938, USA

Received: August 14, 2009; Accepted: October 4, 2009

Abstract

A mutant of green fluorescent protein (GFPmut3*) from
the jellyfish Aequorea victoria was cyclized in vitro and in
vivo by the use of a naturally split intein from the dnaE
gene of Synechocystis species PCC6803 (Ssp). Cyclization
of GFPmut3* was confirmed by amino acid sequencing
and resulted in an increased electrophoretic mobility
compared with the linear GFPmut3*. The circular GFPmut3*
was 5oC more thermostable than the linear form and
significantly more resistant to proteolysis of exopeptidase.
The circular GFPmut3* also displayed increased relative
fluorescence intensity. In addition, chemical stability of
GFPmut3* against GdnHCl revealed more stability of the
circular form compared with the linear form.

Keywords: intein, protein cyclization, purification, green fluorescent protein