Successful protein purification for structural and functional studies typically requires evaluation of multiple expression constructs. Many mutated, truncated or orthologous versions of a protein are required to identify suitable constructs for recombinant expression.  A series of fusion partners may also be investigated for their effects on driving enhanced expression or their capacity to capture and purify the target protein.

We have developed a high-throughput cloning scheme that allows parallel cloning of 12 different constructs or organisms into 8 different vectors using 96 well plate format. We designed a family of eight sequence & ligation-independent cloning (SLIC) vectors with different combinations of protease cleavable N- and C-terminal tags, by replacing multiple cloning site (MCS1) of the commercially available pET Duet vectors. The LIC site in all the vectors of the panel consists of a single restriction site, EcoRI, thus the same gene fragment can be cloned into all these vectors using the same primers. There are 5 family members of such edited pET Duet vectors (in total 40 vectors) with unedited MCS2 and compatible origin of replication, but different antibiotic resistances and copy numbers thus allowing co-expression.

We have successfully used the system to co-express and crystallize the central stalk subunits D and F of yeast V-type ATPase as a proof of principle.