Selected article for: "cleavage site and Genbank accession"
Author: Klaile, Esther; Vorontsova, Olga; Sigmundsson, Kristmundur; Müller, Mario M.; Singer, Bernhard B.; Öfverstedt, Lars-Göran; Svensson, Stina; Skoglund, Ulf; Öbrink, Björn
Title: The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters
  • Document date: 2009_11_16
    • ID: uy2553z7_47
    Snippet: Cloning and expression of Fc-and His-tagged proteins For the rat and human D(1-4)-Fc constructs, rat and human CEACAM1 ectodomains (GenBank accession no. J04963 and X16354) and human IgG Fc (GenBank accession no. BC014258) were amplified (rat CEACAM1, 5-AAGCTTTAGCAGGCAGCAGAGACTATGG-3 and 5-GAATTCA-GAATTTCCTTGTGTTGGATCAGG-3; human CEACAM1, 5-AAGCTTAC-CATGGGGCACCTCTCAGCC-3 and 5-GAATTCAGGTGAGAGGC-CATTTTCTTG-3; and human Fc, .....
    Document: Cloning and expression of Fc-and His-tagged proteins For the rat and human D(1-4)-Fc constructs, rat and human CEACAM1 ectodomains (GenBank accession no. J04963 and X16354) and human IgG Fc (GenBank accession no. BC014258) were amplified (rat CEACAM1, 5-AAGCTTTAGCAGGCAGCAGAGACTATGG-3 and 5-GAATTCA-GAATTTCCTTGTGTTGGATCAGG-3; human CEACAM1, 5-AAGCTTAC-CATGGGGCACCTCTCAGCC-3 and 5-GAATTCAGGTGAGAGGC-CATTTTCTTG-3; and human Fc, 5-GAATTCATGGCACCTGAACTCCT-GGGGGGACC-3 and 5-CTCGAGTCATTTACCCGGAGACAGGGAGA-GGC-3) and ligated sequentially into the HindIII-EcoRI-XhoI sites of pcDNA3.1(+) (Invitrogen). For the rat D(1-4)-His construct, the BsrGI-AgeI cassette of pcDest 40 V5-His (Invitrogen) was replaced with a duplex encoding the TEV protease cleavage site (annealed 5-GTACAAAGCTTAAG-GATCCCGGGCAGCTGGAGAATCTTTATTTTCAGGGCA-3 and 5-CCGGT-GCCCTGAAAATAAAGATTCTCCAGCTGCCCGGGATCCTTAAGCTTT-3). In the resulting vector, the amplified rat CEACAM1 ectodomain (5-AGCTG-GCTAGTTAAGCTATCAACAAGTTTGTAGCCACCATGGAGCTAGCCTCG-GCT-3 and 5-TGATGATGACCGGTGCCCTGAAAATAAAGATTCTCGC-CAGAATTTCCTTGTGTTGG-3) was inserted by homologous recombination. To obtain rat D(2-4)-His, the rat D(1-4)-His vector was amplified (Phusion polymerase; Finnzymes; 5-GGTGACTTGGGCAGTGGT-3 and 5-GCAT-TACAAAAGCCCAACGTC-3, omitting the D1 domain) and self-ligated. Correct sequences and reading frames were verified by sequencing. Recombinant plasmids were transfected into HEK 293 cells, and the respective proteins were allowed to accumulate in serum-free Pro293s-CDM medium (BioWhittaker). Fc fusion proteins were purified by protein A-Sepharose affinity chromatography (HiTrap Protein A HP; GE Healthcare), and Histagged proteins were purified by Ni-NTA affinity chromatography (HisTrap dimer forms for a given A Total , the curve-fit model is composed of the following six coupled differential equations, applied for numerical integration (surface-bound type 1 and type 2 dimers are indicated with R AL and R LA , respectively):

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