Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

• Published in: Acta crystallographica. Section F, Structural biology and crystallization communications Structural biology and crystallization communications, 2010-08, Vol.66 (8), p.871-877
• Main Authors: Gazdag, Emerich Mihai, Cirstea, Ion Cristian, Breitling, Reinhard, Lukeš, Julius, Blankenfeldt, Wulf, Alexandrov, Kirill
• Format: Article
• Language: English
• Subjects: Crystallization; Crystallography, X-Ray; eukaryotic expression systems; Gene Expression; Humans; Leishmania; Leishmania - metabolism; Leishmania tarentolae; Models, Molecular; Protein Structure, Quaternary; recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Structural Communications; Superoxide Dismutase - chemistry; Superoxide Dismutase - genetics; Superoxide Dismutase - isolation & purification; Superoxide Dismutase - metabolism
• ISSN: 1744-3091; 1744-3091; 2053-230X
• DOI: 10.1107/S1744309110019330

The rapid and inexpensive production of high‐quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein‐expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research.