Protein Production
Madison has successfully expressed and purified chicken PLCζ1 in Baculovirus/SF9 system. This protein was found to be active in multiple assays, gave a calcium response when injected into eggs, and has been crystallised.
A detailed description of protein production efforts is below, together with comparisons between human and chicken PLCζ1.
(work performed by Christine Orengo and Vaishali Waman)
Christine and Vaishali (UCL Structural & Molecular Biology) have looked at the similarities of the different orthologs discussed above. Multiple sequence alignment (MSA) and Scorecons analyses indicate that active site is highly conserved across active site residues. Catalytic residues are H311 and H356.
| Active site position (rat:2ISD) | H311 | N312 | E341 | D343 | H356 | E390 | K438 | K440 | S522 | R549 | Y551 |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Human | H | N | E | D | H | E | K | K | S | R | Y |
| Mouse | H | N | E | D | H | E | K | K | S | R | Y |
| Macfa | H | N | E | D | H | E | K | K | S | R | Y |
| Chicken | H | N | E | D | H | E | K | K | S | R | Y |
| Scorecons score | 1 | 1 | 1 | 1 | 1 | 1 | 0.83 | 0.94 | 1 | 1 | 1 |
Note: the Scorecons score is calculated using Valdar program and MSA of a total of 52 mammal species (DOPs: 73.3).
The catalytic domains are compared using structural superposition in Chimera. There is structural conservation of active site region across rat delta (tan), human (orange), macfa (grey), mouse (light blue) and chicken (khaki).
(work performed by Elnur Aliyev and Karl Swann)
Chicken, mouse and human (with chaperone contaminant) PLCζ1 were injected into mouse eggs by Elnur and Karl. All proteins were seen to give a calcium response.
(work performed by Madison Edwards, Eve Carter, Peter Loppnau, Dalia Barsyte-Lovejoy, Magdalena Szewczyk)
Madison has purified full length PLCζ1 from chicken. This has been confirmed to be active by Nuvisan in an IP-one assay. The activity was seen to be similar after the protein was stored at room temperature for 2 h.
Eve has aligned chicken and human PLCζ1 sequences - overall there is 62% identity and 68% identity in the X and Y domains. Overlaying the AlphaFold structures of the X and Y catalytic domains of human and chicken PLCζ1, they are very similar other than the disordered loop region.
Dalia and Magda measured the thermal stability of human, chicken and macfa PLCζ1 in a HiBiT CETSA assay.
(work performed by Madison Edwards and Alma Seitova)
Attempts at expressing full length PLCζ1 from chicken, mouse and macaque in Baculovirus/SF9 system were successful, with results confirmed using an anti-FLAG western blot.
Full-length macaque and mouse PLCζ1s expressed in Baculovirus/SF9 system were purified. These were confirmed by an anti-FLAG western blot. From the SDS-PAGE, it appears that macaque is a monomer whereas mouse is a dimer. The activity of these proteins has been confirmed by Nuvisan using an IP-one assay.
(work performed by Opher Gileadi)
Opher has been using 3 main approaches in E. coli:
- high-throughput split GFP-based screening of engineered PLCζ1 mutants based on alignment of PLCζ1 from different animals
- increasing structural stability with PROSS-identified mutants
- scanning loop deletions to see if the X-Y linker can be made more stable
SDS PAGE of total lysates show PLCζ1 is being expressed, however, when attempting to purify the protein, only GroEL was observed, similar to previous results from Madison.
(work performed by Dalia Barsyte-Lovejoy and Peter Loppnau)
Dalia performed mammalian expression of human PLCζ1 that is codon optimised for humans. Expression appears to be successful, however all bands seen in a western blot are shorter than expected, with the largest appearing to correspond to the loss of the EF hands (this is just through mass on a gel, mass spec has not been performed).
(work performed by Madison Edwards)
Attempts at expressing full length PLCζ1 from chicken, mouse, macaque, cow, pig and horse in E. coli were all unsuccessful, with no desired protein observed.
(work performed by Madison Edwards)
Human MBP-PLCζ1 (1-608 and 105-608) were expressed & purified in the presence of 100 μM CaCl2. Also included 10% glycerol in all buffers. This did not provide any better results than previous experiments.
A test expression of human MBP-PLCζ1 with loop deletions (65-608 Δ302-343) had decent expression but GroEL still present. Attempts to remove GroEL by washing with ATP and an ATP analogue did not work. Other loop deletion constructs had good expression but protein was insoluble.
Expression of different human PLCζ1 constructs (His tagged 1-608, 58-608, 105-608 and 140-608) in mammalian cells was unsuccessful, with no desired protein seen in anti-flag western blots.
Genscript were also unable to purify human PLCζ1 (1-608) from HEK293 cells.
(work performed by Madison Edwards)
After discussion with Michail Nomikos, Madison expressed and purified human PLCζ1 with a His6-MBP tag from E. coli. This gave the best results yet, but the yield was still quite low and contained another band on the gel (image). This unwanted band is thought to be GroEL.
(work performed by Madison Edwards)
Madison Edwards has tried expressing human PLCζ1 with a NusA tag in E. coli. There was a large amount of protein expressed, including the desired construct, however it was insoluble and was found in the cell pellet.
(work performed in the Halabelian group)
As the human PLCζ1 expression was very low in the previous results, a series of additional constructs were cloned using a variety of tags to help improve solubility. Human PLCζ1 was cloned in insect cells with GST, SUMO, N-FLAG, C-His and C-FLAG tags.
Unfortunately, none had good expression of the desired protein.
(work performed in the Halabelian group)
Two human PLCζ1 constructs with Avitag for biotinylation and another with N-terminal histag were cloned in both E. coli and insect cells. Only the insect cells expressing construct showed some weak expression, there was no expression in E. coli.
Expression in insect cells of human PLCζ1 with a biotin tag was scaled up to 4 L, as this was the most promising construct from the test expressions. A faint band at the right size was visible in the gel after TALON purification, but size exclusion chromatography was not successful, with many faint bands being collected