For your current PSI-2 target ORFs, boundaries defined by domain family analysis and some constructs are generated per target to optimize the means to get a successful outcome. Our permutation strategy will depend on extension with the N or C-termini depending on secondary structure, length restrictions or experimental data. When possible however, we attempted cloning and expression of the original full-length-target sequence. A lot of targets represent the MSCG assigned Pfam groups with approximately 25% of the target group composed of biomedical targets from pathogens. For that expressed target group, we observed a total rate of success of ~25% for generation associated with an expression clone that produced a soluble protein product sufficient for crystallographic studies.
In order to meet the fabrication goals for PSI-2, we apply a wide 96-well-plate HTP technology to come up with clones and express soluble proteins. Our pipelines use pMCSG7 because the primary expression vector along with a maltose-binding protein (MBP) fusion vector to get a “salvage” technique for proteins that express well in pMCSG7 but show low solubility. Expression clones that produce insoluble proteins are directed to Level 2 processing (see Figure). The developmental goal is to address solubility problems using HTP approaches. Criteria for entry to the salvage loop would include: deficit of a soluble orthologues, poor diffraction quality crystals, or high target priority due to biomedical impact. This tiered strategy leverages our efficient and cost-effective parallel processes suitable for mass production of proteins and protein fragments in E. coli.
Coding regions are amplified using primers made with the Express Primer tool or domain-specific primer design tools. All primers contain ligation-independent cloning sites compatible with multiple vectors. Affinity tags having a TEV (tobacco etch virus) protease cleavage site are fused to any or all proteins to facilitate their purification or capture. The main steps on the process — PCR gene amplification, testing for protein expression and solubility — are conducted in 96-well-plate format. Denaturing PAGE analysis of proteins is performed in a high-density gel format.
In the event the PSI pilot centers were formed, ligation-independent cloning (LIC) offered a stylish technology adaptable for robotic cloning, but existing vectors weren’t suited to automated purification of proteins for crystallization. We developed a list of superior LIC vectors tailored tailored for this purpose. The vector, pMCSG7, encodes a His6-tag followed by a spacer and a TEV protease cleavage site that overlaps while using LIC site. This design puts the TEV site near to the start of the cloned native protein. Only the three-amino-acid-sequence SerAsnAla
If the PSI pilot centers were formed, ligation-independent cloning (LIC) offered a lovely technology adaptable for robotic cloning, but existing vectors just weren’t made for automated purification of proteins for crystallization. We developed a pair of superior LIC vectors tailored designed for this purpose. The vector, pMCSG7, encodes a His6-tag then a spacer and also a TEV protease cleavage site that overlaps using the LIC site. This design puts the TEV site close to the start of the cloned native protein. The three-amino-acid-sequence SerAsnAla (SNA) is added to the protein after protease cleavage.