Abstract
In this chapter, we present methods for adapting the eukaryotic cell-free expression system based on Leishmania tarentolae to high-throughput analysis of protein interactions. Specifically, we present a lysate optimization technique that minimizes the amount of unwanted premature termination products while balancing protein expression level and protein aggregation. Finally, we present methods for adapting the Leishmania cell-free system to the AlphaLISA-based protein interaction assay.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gagoski D et al (2016) Performance benchmarking of four cell-free protein expression systems. Biotechnol Bioeng 113(2):292–300
Johnston WA, Alexandrov K (2014) Production of eukaryotic cell-free lysate from Leishmania tarentolae. In: Alexandrov K, Johnston WA (eds) Cell-free protein synthesis: methods and protocols. Humana Press, Totowa, NJ, pp 1–15
Mureev S et al (2009) Species-independent translational leaders facilitate cell-free expression. Nat Biotechnol 27(8):747–752
Ullman EF et al (1994) Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci 91(12):5426–5430
Yasgar A et al (2016). AlphaScreen-based assays: ultra-high-throughput screening for small-molecule inhibitors of challenging enzymes and proteinprotein interactions. In W. P. Janzen (Ed.), High Throughput Screening: Methods and Protocols (Vol. 1439, pp. 77–98). New York, NY: Springer
Banaszynski LA, Liu CW, Wandless TJ (2005) Characterization of the FKBP⊙ Rapamycin⊙ FRB Ternary Complex. J Am Chem Soc 127(13):4715–4721
Chen J et al (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci 92(11):4947–4951
Fridy PC et al (2014) A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods 11(12):1253–1260
PerkinElmer (2015) Anti-GFP AlphaLISA protocol. Available from: https://www.perkinelmer.com/Content/TDLotSheet/AL133C_2059866.pdf
Hunter DJB, Bhumkar A, Giles N, Sierecki E, Gambin Y (2018) Unexpected instabilities explain batch-to-batch variability in cell-free protein expression systems. Biotechnol Bioeng 115(8):1904–1914
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Johnston, W.A., Moradi, S.V., Alexandrov, K. (2019). Adaption of the Leishmania Cell-Free Expression System to High-Throughput Analysis of Protein Interactions. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9624-7_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9623-0
Online ISBN: 978-1-4939-9624-7
eBook Packages: Springer Protocols