Table 1.
Synthetic oligonucleotides used to amplify Aα-chain fragments and for site-directed and deletion mutants of the α-chain
Restriction endonuclease sites are underlined. The nucleotides in boldface indicate the location of the desired mutation. F corresponds to forward primer; R corresponds to reverse primer.
| Expression constructs | Primer name | Primer sequence |
|---|---|---|
| Truncated proteins | Aα-1F | CGG GGA TCC GCA GAT AGT GGT GAA GGT G |
| Aα-232F | GAC GGA TCC TTA ACA GAC ATG CCG CAG ATG AG | |
| Aα-315F | CGG GGA TCC TGG AAC TCT GGG AGC TCT GG | |
| Aα-367F | CGG GGA TCC TGG CAC TCT GAA TCT GGA AG | |
| Aα-283R | CGG AAG CTT AGG TCC AGA GCT CCC AGA GTT C | |
| Aα-574R | CGG AAG CTT TTA GTC TCC TCT GTT GTA ACT CGT G | |
| Aα-625R | CGG AAG CTT TTA GGG GGA CAG GGA AGC CTT C | |
| Deletion mutagenesis | Aα-329F | GCG CTG ATA TCG GAA ACC AAA ACC CTG GGA G |
| Aα-341F | GCG CTG ATA TCG GTA GTA CCG GAA CCT GG | |
| Aα-344F | GCG CTG ATA TCT GGA ATC CTG GCA GCT CTG | |
| Aα-373F | CGC TGA TAT CGG AAG TTT TAG GCC AGA TAG | |
| Aα-286R | ATC TCG GTT TCC AGT ACT TCC AG | |
| Aα-315R | ATC CCA GCT TCC AGT ACT TCC AG | |
| Aα-331R | ATC GTC CCC AGG GTT TTG GTT CTT | |
| Aα-341R | ATC CCA GGT TCC GGT ACT ACC AG | |
| Site-directed mutagenesis | Aα-S317PF | GCTGGAACCCTGGGAGCTCTGGAAC |
| Aα-S317PR | GTTCCAGAGCTCCCAGGGTTCCAGC | |
| Aα-T322PF | TGGGAGCTCTGGACCTGGAAGTACTGGAAAC | |
| Aα-T322PR | GTTTCCAGTACTTCCAGGTCCAGAGCTCCCA | |
| ClfB mutant | N526F | GTT GGA ATA ATG AGG CTG TTG TAC GTT ATG GTG G |
| N526R | CCA CCA TAA CGT ACA ACA GCC TCA TTA TTC CAA C |