A modified IDR-1002 wasynthesizeded by incorporating a C-terminal cysteine IDR-1002CC) to allow the presence of a thiol group for biotinylation of the peptide.
Transcription templates were generated by PCR, in most cases incorporating a C-terminal V5 tag via the reverse primer, and RNA produced as described previously [ 11].
CD80 and CD86 coding sequences were amplified by PCR from DC2.4 cell cDNA incorporating a C-terminal HA tag, as well as EcoRI and BglII restriction sites for insertion into the pCAGGS expression vector.
A DNA fragment containing residues 1 440 of HsrTSγ (IMAGE clone: 3454185) was subcloned into the pNIC28-Bsa4 vector (GenBank accession EF198106) incorporating an N-terminal TEV-cleavable His6-tag.
We cloned the NEMOwCcc construct into the pCDNA3.1 vector, incorporating an N-terminal FLAG tag for co-immunoprecipitation (co-IP) experiments.
DNA fragments encoding the kinase domain of human GAK (residues 14 351 for the co-crystal structure or 12 347 for the apo structure) were subcloned into either the pNIC-H102 or pNIC28-Bsa4 vector, both incorporating an N-terminal TEV (tobacco etch virus -cleavable His6 tag.
DNA fragments encoding hMAT1A (residues 16 395; NCBI GI number 4557737), hMAT2A (residues 1 395; NCBI GI number 5174529) and hMAT2B (residues 14 323; NCBI GI number 37182512) were subcloned into the pNIC28-Bsa4 vector incorporating an N-terminal TEV (tobacco etch virus -cleavable His6 tag.
MaviP35 was also amplified with oligonucleotides 3 and 5 and cloned the same way into pGALL- HIS3), to incorpGALL- HIS3rboxyl tormincorporateag.
We demonstrate two expression systems used to generate Ig fusion proteins, the first utilised a commercial vector to incorporate an amino terminal leader sequence and carboxy terminal Ig domains.
