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pYD5 plasmid vector酵母表面展示质粒载体
pYD1 is the parental vector with the surface expression cassette located C-terminal to the Aga2p yeast membrane-associated protein and pYD5 is the modified vector in the reverse configuration. Figure 1B shows the sequence of the Aga2 signal peptide and how an NheI site was added by a silent mutation just proximal to the last three residues of the signal peptide before the cleavage site. Figure 1C shows how an EcoRI site was added downstream from the NheI site in pYD1 leading to pYD5. This permits cloning in scFv inserts having the sequence ASVLA-(scFv)-EF into pYD5, where AS and EF are NheI and EcoRI sites. We used a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and followed the manufacturer's instructions: sense primer, (5′ TCA ATA TTT TCT GTT ATT GCT AGC GTT TTA GCA CAG GAA CTG ACA ACT ATA TGC 3′); antisense primer, (5′ GCA TAT AGT TGT CAG TTC CTG TGC TAA AAC GCT AGC AAT AAC AGA AAA TAT TGA 3′). We defined this mutated construct as pYD2. The two fragments, NheI–VLA–EcoRI–V5–(G4S)2–BamHI, sense primer carrying NheI and EcoRI, (5′ CTA GCT AGC GTT TTA GCA GAA TTC GGT AAG CCT ATC CCT AAC CCT 3′), antisense primer carrying BamHI, (5′ CGC GGA TCC ACC ACC ACC AGA ACC ACC ACC ACC CGT AGA ATC GAG ACC GAG GAG 3′), BglII–(G4S)–Aga2–stop–PmeI–HindIII, sense primer carrying BglII, (5′ GGA AGA TCT GGT GGT GGT GGT TCT CAG GAA CTG ACA ACT ATA TGC 3′), antisense primer carrying PmeI and HindIII, (5′ CCC AAG CTTGTTTAAAC TCA AAA AAC ATA CTG TGT GTT TAT GGG 3′) were PCR amplified with high fidelity cloned pfu DNA polymerase. They were digested with NheI–BamHI and BglII–HindIII, respectively. The two fragments were co-cloned into pET17b between NheI and HindIII sites. The construct was confirmed by sequencing and in-frame checking by expression in E.coli BL21 (DE3) pLysS and western blot analysis with goat anti-mouse IgG-HRP. The insert was then subcloned into pYD2 between the NheI and PmeI sites. We defined the final construct as pYD5.
Supplier来源:BioVector NTCC Inc.
TEL电话:400-800-2947
Website网址: http://www.biovector.net
pYD1 is the parental vector with the surface expression cassette located C-terminal to the Aga2p yeast membrane-associated protein and pYD5 is the modified vector in the reverse configuration. Figure 1B shows the sequence of the Aga2 signal peptide and how an NheI site was added by a silent mutation just proximal to the last three residues of the signal peptide before the cleavage site. Figure 1C shows how an EcoRI site was added downstream from the NheI site in pYD1 leading to pYD5. This permits cloning in scFv inserts having the sequence ASVLA-(scFv)-EF into pYD5, where AS and EF are NheI and EcoRI sites. We used a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and followed the manufacturer's instructions: sense primer, (5′ TCA ATA TTT TCT GTT ATT GCT AGC GTT TTA GCA CAG GAA CTG ACA ACT ATA TGC 3′); antisense primer, (5′ GCA TAT AGT TGT CAG TTC CTG TGC TAA AAC GCT AGC AAT AAC AGA AAA TAT TGA 3′). We defined this mutated construct as pYD2. The two fragments, NheI–VLA–EcoRI–V5–(G4S)2–BamHI, sense primer carrying NheI and EcoRI, (5′ CTA GCT AGC GTT TTA GCA GAA TTC GGT AAG CCT ATC CCT AAC CCT 3′), antisense primer carrying BamHI, (5′ CGC GGA TCC ACC ACC ACC AGA ACC ACC ACC ACC CGT AGA ATC GAG ACC GAG GAG 3′), BglII–(G4S)–Aga2–stop–PmeI–HindIII, sense primer carrying BglII, (5′ GGA AGA TCT GGT GGT GGT GGT TCT CAG GAA CTG ACA ACT ATA TGC 3′), antisense primer carrying PmeI and HindIII, (5′ CCC AAG CTTGTTTAAAC TCA AAA AAC ATA CTG TGT GTT TAT GGG 3′) were PCR amplified with high fidelity cloned pfu DNA polymerase. They were digested with NheI–BamHI and BglII–HindIII, respectively. The two fragments were co-cloned into pET17b between NheI and HindIII sites. The construct was confirmed by sequencing and in-frame checking by expression in E.coli BL21 (DE3) pLysS and western blot analysis with goat anti-mouse IgG-HRP. The insert was then subcloned into pYD2 between the NheI and PmeI sites. We defined the final construct as pYD5.
Supplier来源:BioVector NTCC Inc.
TEL电话:400-800-2947
Website网址: http://www.biovector.net
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