The peptide was (1) attached to the C-terminus end of the cellulose binding domain (CBD), (2) inserted in the glycosylated linker region, (3) added after a truncated form of EGI lacking the CBD and a small part of the linker.
The culture dishes were then inserted in the UV cross-linker (UVP) and the lids were removed.
Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells.
The 10 amino acid sequence identified in AHNAK-N was inserted into the linker (Figure 1B), and fusion constructs with and without the AHNAK motif were co-expressed with CAPN3 or its proteolytically inactive mutant CAPN3C129S.
Since we wanted to use the same tag for each targeting vector, EGFP was inserted into the linker region that separates PHM from PAL in PAM1; 55 amino acids of the linker region were replaced by EGFP, with no deleterious effect on either enzymatic activity [ 22].
To increase the thermostability of the BGL-CBD, a flexible peptide linker containing (ProProPro) was inserted in the middle of the BGL-CBD, which often conferred the necessary flexibility for the individual proteins to maintain their functionalities.
The E. coli uidA+ gene, encoding β-glucuronidase, was amplified by PCR and, using standard in vitro molecular cloning techniques, the resulting 1.8 kbp uidA+ coding sequence was inserted in-frame into the 21 bp crossover linker sequence of pHyqΔRI7 yielding plasmid pHyqRU5 (Table 1; Methods).
The cDNA encoding GUSh was inserted in frame with the scFv separated by a 15-amino-acid linker segment containing (Gly4Ser)2 flanked on both sides by three alanine residues, encoded by the NotI sites.
In this new class of analogs, we inserted an ethylene moiety in the linker and used indole as a part of the Y-shaped cap group.
