Expression of protein complexes using multiple Escherichia coli protein co-expression systems: A benchmarking study

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• 作者：Didier Busso^a ; ¹ ; Yoav Peleg^b ; ¹ ; Tatjana Heidebrecht^c ; Christophe Romier^a ; Yossi Jacobovitch^b ; Ada Dantes^b ; Loubna Salim^a ; Edouard Troesch^a ; Anja Schuetz^d ; Udo Heinemann^d ; ^e ; Gert E. Folkers^f ; Arie Geerlof^g ; ² ; Matthias Wilmanns^g ; Andrea Polewacz^h ; Claudia Quedenau^h ; Konrad Bü ; ssow^h ; ⁱ ; Rachel Adamson^j ; Elena Blagova^k ; Julia Walton^k ; Jared L. Cartwright^j ; Louise E. Bird^l ; ³ ; Raymond J. Owens^l ; ³ ; Nick S. Berrow^l ; ⁴ ; Keith S. Wilson^k ; Joel L. Sussman^b ; ^m ; Anastassis Perrakis^c ; Patrick H.N. Celie^c ; ^{p.celie@nki.nl"" rel=""nofollow}
• 关键词：Escherichia coli ; Co-expression ; Cloning strategies ; Enzyme-free cloning ; Gateway™ ; In-Fusion™ ; LIC ; Restriction-free cloning
• 刊名：Journal of Structural Biology
• 出版年：2011
• 出版时间：August 2011
• 年：2011
• 卷：175
• 期：2
• 页码：159-170
• 全文大小：1237 K

文摘

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no ‘silver bullet’ approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.