The influences of the N- and C- terminal addition of FLAG/(His 6 tag spaced by pentaglycine sequence and the sequentially accumulative deletions of N-glycosylation sites within hFasLECD were investigated.
The study demonstrated that the C-terminal addition of the poly-histidine tag to SjGST increased the metal affinity of the enzyme to the Co-TANOL gel under both native and denaturing conditions and to the Ni-NTA gel under denaturing conditions, whereas the enzymatic activity and antibody-binding ability were not affected.
As judged from SDS PAGE analysis of the culture supernatant, the N-terminal addition of FLAG-(Gly)5 tag and the deletion of single N-glycosylation site via N184Q mutation increased the secretion level of the product.
Fusion protein constructs were made by subcloning DNA fragments of interest into the 35S puc18-spGFP6 vector using restriction enzymes BamHI/ NheI for N-terminal GFP addition, and restriction enzymes XbaI/ SpeI for the C-terminal addition of the RbcS 35S puc18-spGFP6 region (UTR) (M. Tegeder, unpublished data) (New England BioLabs, Ipswich, MA).
This avoids the N-terminal addition of amino acids derived from the junction of the IRES to the coding sequence of interest.
The C-terminal addition of loxP and the HIV-1 Rev-NES had no significant effect on the function of wild-type Upf3p (data not shown), indicating that the NMD phenotype of the mutant Upf3-loxP and Upf3-loxP-Rev proteins would be due solely to their amino acid substitutions in NES-A.
The HASPs are dually acylated by the N-terminal addition of myristate and palmitate, co- and post-translational modifications that are essential for plasma membrane trafficking (Denny et al., 2000).
Receptor function is not altered by the N-terminal addition of this tag (Hurley et al., 1999).
These results suggest that the C-terminal addition of two Glu residues to β-conformer peptide DFVIFLDVKHFSPEDLT is sufficient to affect the secondary structure.
The N-terminal addition of a laminin-derived LamininNT-EGF supra-domain or a frizzled-CRD domain to a single netrin domain is the most parsimonious explanation, because the C-terminal addition of a netrin domain to a LamininNT-EGF supra-domain or a frizzled-CRD domain would require an additional loss of the C-terminal domains of the "hosts" laminins and frizzled.
It was not clear whether folding of the chemokine would tolerate the N- or C-terminal addition of biotinylation sequences, so constructs tagged at either end were tried.
