CloningThe main advantage of having a capacity of this magnitude is the ability to generate multiple constructs of the target proteins rather than focusing only on the full-length versions. A “multi-construct approach” based on protein domains of interest dramatically increases the likelihood of obtaining soluble and correctly folded protein at the end of the pipeline1. Although originally from a structural biology setting, we have found that this approach is amenable to other fields of research as well. |
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All cloning is performed in 96-well format using ligation-independent cloning (LIC) technology in Escherichia coli 2,3. Usually, all targets are fused with an N-terminal hexa-histidine tag to aid in downstream purification and a TEV-protease cleavage site for optional tag removal. The main vector in use is pNIC28-Bsa4 (GenBank accession EF198106), developed by Opher Gileadi at SGC Oxford. We use a RecA- strain of E. coli for the initial cloning work and plasmid production followed by a re-transformation of the construct into an expression strain (typically, a T1-phage resistant BL21-DE3 Rosetta strain developed in house). All clones will be put through a small-scale analytical expression screen to assess the likelihood of successful large-scale protein expression and purification. Basically, we use 96-deep well blocks to grow all cultures at a volume of 1 mL Terrific Broth. Once the optical density at 600nm reaches 2-3, the temperature will be lowered from 37ºC to 18ºC and protein production induced with IPTG. After over-night cultivation, we take out a small sample from the liquid culture and verify the presence of target protein using SDS-PAGE. We also continue to perform cell lysis, clarification and micro-scale immobilized metal-ion affinity chromatography (IMAC) to assess the levels of soluble and purifiable target protein from each clone.
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