Despite the large body of knowledge accumulated on recombinant protein expression, production, primarily of eukaryotic proteins, remains a challenge. The biggest obstacle is in obtaining large amounts of a given protein in a correctly folded form. Several strategies are being used to increase both yields and solubility. These include expression with fusion proteins, co-expression with molecular chaperones or a protein partner, and use of multiple constructs for each protein. Any given method may help to increase expression and solubility for a given protein, but often more than one rescue strategy should be tried. To perform several different rescue strategies on multiple proteins, high throughout (HTP) methodologies are applied. This chapter presents HTP methodologies for DNA cloning in multiple expression vectors and expression screening to identify clones capable of producing soluble proteins.
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Acknowledgments
The authors acknowledge the support of the Divadol Foundation, the Newman Foundation, The Israel Ministry of Science, Culture and Sport grant for the ISPC, and the European Commission Sixth Framework Research and Technological Development Programme “SPINE2-COMPLEXES” Project under contract No. 031220. The authors thank Joel L. Sussman and Israel Silman for their comments on the manuscript.
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Peleg, Y., Unger, T. (2008). Application of High-Throughput Methodologies to the Expression of Recombinant Proteins in E. coli . In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_12
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