A systematic investigation of the stability of green fluorescent protein fusion proteins.

Monika Janczak; Michał Bukowski; Andrzej Górecki; Grzegorz Dubin; Adam Dubin; Benedykt Wladyka

Monika Janczak Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.;
Michał Bukowski Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.;
Andrzej Górecki Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.;
Grzegorz Dubin Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.;
Adam Dubin Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.;
Benedykt Wladyka Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland.;

Abstract

X-ray crystallography provides important insights into structure-function relationship in biomolecules. However, protein crystals are usually hard to obtain which hinders our understanding of multiple important processes. Crystallization requires large amount of protein sample, whereas recombinant proteins are often unstable or insoluble. Green fluorescent protein (GFP) fusion is one of the approaches to increase protein synthesis, solubility and stability, facilitating crystallization. In this study we analyze the influence of the linker length, composition and the position of GFP relative to the fusion partner on the fusion protein production and stability. To this end, multiple constructs of enzymatically impaired variant of PemKSa toxin from Staphylococcus aureus CH91 fused to GFP were generated. Fusion protein production in Escherichia coli was evaluated. The proteins were purified and their stability tested. PemKSa-α14aa-GFP fusion provided best production and stability. Obtained results demonstrate the importance of optimization of fusion protein construct, including linker selection and the order of fusion partners, in obtaining high quantities of stable protein for crystallization.