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bioRxiv · 10.1101/2021.08.10.455798

pOPIN-GG: A resource for modular assembly in protein expression vectors

Bentham, A. R.·Youles, M.·Mendel, M. N.·Varden, F. A.·De la Concepcion, J. C.·Banfield, M. J.
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Abstract

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials. HighlightsO_LIpOPIN-GG expression vectors allow for modular cloning enabling rapid screening of purification and solubility tags at no loss of expression compared to previous vectors. C_LIO_LICloning into the pOPIN-GG vectors can be performed from PCR products or from level 0 vectors containing the required parts. C_LIO_LISeveral vectors with different resistances and origins of replication have been generated allowing the effective co-expression and purification of protein complexes. C_LIO_LIAll pOPIN-GG vectors generated here are available on Addgene, as well as level 0 acceptors and tags. C_LI

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Bentham, A. R., Youles, M., Mendel, M. N., Varden, F. A., De la Concepcion, J. C., Banfield, M. J.. 2021-08-10. pOPIN-GG: A resource for modular assembly in protein expression vectors. https://doi.org/10.1101/2021.08.10.455798

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