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.
journal_name
Acta Biochim Poljournal_title
Acta biochimica Polonicaauthors
Janczak M,Bukowski M,Górecki A,Dubin G,Dubin A,Wladyka Bdoi
10.18388/abp.2015_1026subject
Has Abstractpub_date
2015-01-01 00:00:00pages
407-11issue
3eissn
0001-527Xissn
1734-154Xpii
2015_1026journal_volume
62pub_type
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