Protein Targets

Image Credits: https://www.idtdna.com/pages/education/decoded/article/oligo-synthesis-why-idt-leads-the-oligo-industry

Construct: WT Nb

Overall Design: Wild-type nanobodies are expressed in pET-29b plasmid. pET-29b plamid has T7 promoter and lac operon to control the expression, and histidine tag for purification.

Construct: Targets

Overall Design: pET-42XH is used to express target proteins. pET-42XH system has a Glutathione-S-transferase tag for protein purification. Besides, we added Avitag, and an HRC-3C site to the protein sequence. Avitag can be biotinylated, which allows the protein to be coated on the surface of the microfluidic device. HRC-3C site is recognized by the HRC-3C protease, which can be used to remove the GST tag after purification.

MAGEB1

Expected Size: 33kb

Tau2

Expected Size: 45kb

Nb-B

Expected Size: 32kb

Induction

All proteins are induced with 0.4mM IPTG at 30 ˚C for 4 hours. Target proteins are purified by GST column and WT nanobodies are purified by Ni-NTA columns. Constructs for proteins (CTLA4, MB1, tau, SSPS1, NbA, and NbB) are all inducable but HB2 was unsuccessful. The three proteins on the left were induced but remain insoluble.

Credit: Chi Zhang

Page updated

Report abuse