Protein cyclization enhanced thermostability and exopeptidase-resistance of green fluorescent protein.
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 5 degrees C 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.
Zhao
Zhonglin
Z
Biotechnology Research Institutee, Chinese Academy of Agricultural Sciencess, Beijing 100081, PR China.
Ma
Xin
X
Li
Liang
L
Zhang
Wei
W
Ping
Shuzhen
S
Xu
Ming-Qun
MQ
Lin
Min
M
D015153:Blotting, Western; D003500:Cyclization; D004247:DNA / Q000737:chemistry / Q000235:genetics; D020689:Exopeptidases / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047668:Inteins; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D046939:Synechocystis / Q000737:chemistry* / Q000235:genetics
C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D004247:DNA; D020689:Exopeptidases