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Cloning, expression, crystallization and preliminary X‐ray studies of a superfolder GFP fusion of cyanobacterial Psb32
Author(s) -
Liauw Pasqual,
Kannchen Daniela,
Gasper Raphael,
DyczmonsNowaczyk Nina,
Nowaczyk Marc M.,
Hofmann Eckhard
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x15003970
Subject(s) - green fluorescent protein , fusion protein , cloning (programming) , fusion , arabidopsis thaliana , thermophile , expression vector , fluorescence , recombinant dna , crystallization , chemistry , biology , computational biology , microbiology and biotechnology , biochemistry , gene , physics , computer science , optics , linguistics , philosophy , enzyme , organic chemistry , mutant , programming language
A fusion of Psb32 from the thermophilic cyanobacterium Thermosynechococcus elongatus BP‐1 ( Te Psb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space group P 6 1 22. A full data set extending to 2.3 Å resolution was collected at the Swiss Light Source. The phase problem could be solved by using only the sfGFP fusion partner or by using GFP and At TLP18.3 from Arabidopsis thaliana as search models. Based on this expression construct, a versatile library of 24 vectors combining four different superfolder GFP variants and three affinity tags was generated to facilitate expression and screening of fluorescent fusion proteins.