Part:BBa_K4190020

GLP-1 Receptor Gene Insert in E. Coli

His-tagged GLP-1 R coding region to be inserted into a plasmid via GGA and expressed in E. coli. Expresses only surface binding site.

Usage and Biology

The native GLP-1 receptor (GLP1-R) is found on the surface membrane of pancreatic cells and is 463 amino acids in size [1]. The binding domain of the receptor is 365 base pairs and feasible for production in a recombinant vector. The crystal structure of binding between Ex-4 and the GLP1-R allows for a truncated GLP1-R (GLP1-RΔ) to be an effective alternative for testing of binding in the respective binding pocket [2]. The GLP1-R gene block of 505 bases will be inserted into the pET28 backbone via golden gate and expressed in BL21 E. coli cells. The GLP1-R protein will be purified for use during Ex-4 binding affinity tests. Abinding affinity test between the truncated GLP1-R and Ex-4 via an octet machine will allow for the secondary folding of the Ex-4 and functionality to be qualitatively deduced. The binding affinity of manufactured Ex-4 is very strong at 6 nmol [3].

Restriction Enzyme Sites

For both E. coli and S. cerevisiae, we used the type IIS restriction enzyme, BsaI, on our insert. This restriction enzyme binds to the sequence GGTCTC and cuts after the first and fifth non-specific nucleotides, which results in “sticky ends” downstream of the binding site [4]. The restriction enzyme site was present on the top and bottom strands respectively in the five prime to three prime direction. The desired insert sequence was constructed to allow scarless integration into pET:28-GFP.

Histidine Tag

After the start codon for our desired gene code, the first element is the histidine tag (his-tag). Our his-tag is made up of six histidine amino acids. When translated, the his-tag will be connected to the N-terminus of our Exendin-4 (Ex-4) protein. This allows us to verify that our desired protein was expressed through protein purification [5].

We used a ten amino acid linker of alternating glycine and serines to distance our his-tag from the Ex-4 polypeptide, and enhance his-tag exposure for protein purification.

For the gene insert containing the sequence to produce the GLP-1 receptor in E. coli, the enterokinase cut site was replaced with a TEV protease cut site. This was done because our lab was given TEV protease by the Dubois Lab at UCSC.

Codon Optimized Protein Sequence

A codon optimized sequence for the GLP1 receptor was also added to a separate gene insert for binding affinity testing. Since this gene insert contained a TEV protease cut site rather than an enterokinase cut site, an alanine was coded for in between the cut site and the GLP1 receptor sequence. This is because TEV protease will only cut before serine, glycine, alanine, methionine, cysteine, or histidine [6].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 140
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 11
  Illegal BsaI.rc site found at 238
  

References

[1] “Glucagon Like Peptide 1 Receptor - an overview | ScienceDirect Topics.” https://www.sciencedirect.com/topics/medicine-and-dentistry/glucagonlike- peptide-1-receptor/ (accessed Aug. 28, 2022).

[2] “GLP1R glucagon like peptide 1 receptor [Homo sapiens (human)] - Gene - NCBI.” https://www.ncbi.nlm.nih.gov/gene/2740 (accessed Aug. 28, 2022).

[3] M. K. K. Yap and N. Misuan, “Exendin-4 from Heloderma suspectum venom: From discovery to its latest application as type II diabetes combatant,” Basic & Clinical Pharmacology & Toxicology, vol. 124, no. 5, pp. 513–527, 2019, doi: 10.1111/bcpt.13169.

[4] J. H. Lee, H. J. Won, E.-S. Oh, M.-H. Oh, and J. H. Jung, “Golden Gate Cloning- Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants,” Front. Plant Sci., vol. 11, p. 559365, Oct. 2020, doi: 10.3389/fpls.2020.559365.

[5] “His-Tag Purification | Chromatography Media, Columns and Kits | Bio- Rad.” https://www.bio-rad.com/featured/en/his-tag-purification.html (accessed Aug. 27, 2022).

[6] “TEV Protease | NEB.” https://www.neb.com/products/p8112-tev-protease (accessed Aug. 27, 2022).