PARTS

The protein is N-terminally fused to a subunit of the α-agglutinin receptor AGA-2P and linked to AGA-1P by disulfide bridges. AGA-1P is anchored at the yeast cell surface. The fusion protein also contains a linker peptide and an Xpress epitope N-terminally as well as a V5 epitope and a his6-tag C-terminally. The expression is controlled by the promoter GAL1, enabling inducible expression by adding Galactose.Additionally, the vector contains an ori for the replication in E. coli as well as CEN6/ARS4 for replication in S. cerivisiae. The vector contains an ampicillin resistance gene and the gene TRP1 enabling tryptophane synthesis in auxotrophic yeast strains. 1

Stream upwards and downwards of LanM pYD1-fw and pYD1-rev are located, enabling rechecking of the correct insert size.

The pET28a-mostcomm-Strep-17x-LCI expression system is used to express the lanthanoid binding peptide ”most common standard tag” fused with a Strep-tag and 17x-LCI-anchor in E. coli – mostcomm-Strep-17x-LCI.

The protein N-terminally contains a lanthanoid binding sequence “most common standard tag” (“MC”) 4,5, one of the peptides we evaluated for application in our experiments and found to selectively bind significant amounts of neodymium (for more information see “Experiments and Procedures”). This sequence is followed by a Strep-tag 6 that consist of 8 amino acids which originate from the bacterium Streptomyces avidinii. The Strep-tag has a strong affinity for Strep-Tactin, which is used for purification of the fusion proteins or in our case peptides via affinity chromatography. Also, the Strep-tag has a diminished propensity to be protonated at low pH rather than a His-Tag which was a considerable factor for our application. The peptide is fused with a LCI-anchor 7 on a 17x linker to guarantee flexibility and freedom of the binding sequence with the fusion peptide immobilized on polystyrene – as was in our application 8.