Expression, purification and crystallization of cysteine-rich human protein muskelin in Escherichia coli.
The increasing interest in the structural arrangements and functional interdependencies of individual modules within large multidomain proteins requires the development of new methods allowing efficient production and purification of large human proteins. Heterologous expression in bacteria is still the most convenient and widely-used approach. However, most of the existing tools are not well suited to expression of cysteine-rich proteins in a native-like soluble form, and with the increasing protein size refolding may result in obtaining non-native conformations or improper disulfide bridging pattern. Here, we present an efficient method of expression and purification of muskelin, a large, multidomain, cysteine-rich eukaryotic protein involved in cell adhesion and regulation of cytoskeleton dynamics. Using a broad range of purification and solubility tags, expression strains and conditions we optimized the procedure to acquire a natively folded protein of crystallization-scale quantity and purity. The correct protein conformation and disulfide bonding were anticipated from the results of circular dichroism spectra and Ellman's assay. Successful crystallization trials are a step towards muskelin crystal-structure determination, while the optimized expression and purification procedure can easily be applied to produce other eukaryotic proteins in the bacterial expression system.
Kiedzierska
Adriana
A
Faculty of Biotechnology, Laboratory of Protein Engineering, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
Czepczynska
Hanna
H
Smietana
Katarzyna
K
Otlewski
Jacek
J
D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D015815:Cell Adhesion Molecules; D047908:Intracellular Signaling Peptides and Proteins; C503019:MKLN1 protein, human; D011994:Recombinant Proteins