A selection of Golden Gate vectors to simplify recombinant protein production inEscherichia coli

Abstract

In this work a total of 22 Escherichia coli vectors have been created to allow the simple cloning (via Golden Gate) of the same DNA insert into multiple plasmids. This approach rapidly generates not only his, twin-strep and avi tagged constructs but also a variety of fusion proteins including the commonly used SUMO, MBP, GST and sfGFP. Each vector contains two BsaI sites allowing the Golden Gate cloning of inserts under the control of the powerful T7 promoter. This allow for construct expression in the standard protein production strain of E. coli BL21[DE3]. To further simplify use all vectors encode kanamycin resistance. We have evaluated the vectors using a test super folder GFP insert and found that using the Golden Gate process allows cloning efficiencies of greater than 90% to be routinely obtained. This allows the cloning and evaluation of many vector insert combinations to be achieved in a short time frame. The plasmid vector set described herein should prove useful to any investigator who has to routinely evaluate numerous protein expression constructs. These vectors are freely available on request or via Addgene.

Crystallographic fragment screening delivers diverse chemical scaffolds for Zika virus NS2B-NS3 protease inhibitor development

“…The construct was created by using a synthetic E. coli codon optimised gene ZIKV sequence as template (accession YP 009227202.1). Golden gate cloning was used to insert this into the pNIC-HIS6-GST-TEV-GG vector 21 . The final construct contains a GST fusion with a TEV site followed by the NS2B peptide (residues 45-89 aa).…”

Crystallographic fragment screen of Enterovirus D68 3C protease and iterative design of lead-like compounds using structure-guided expansions

“…A synthetic gene, codon optimised for expression from E.coli, corresponding to residues 1549-1731 of the Enterovirus D68 polyprotein (Uniprot id: Q68T42.1) was ordered from TWIST Bioscience. TThe gene was cloned using the Golden Gate method [15] into a pNIC vector (https://www.addgene.org/215810/) with a non-cleavable C-terminal hexaHIS tag (bold -below). The final construct sequence is:…”