TABLE 1.
Primer pairs used for cloning and mutagenesis
The underlined sequence indicates the restriction sites used for cloning. Boldface sequences indicate site-directed mutations. A conservative mutation was made within GLP-1 to remove an internal EcoRI restriction site. Boldface and underlined text indicates a stop codon.
| Fusion construct | Oligonucleotide sequence pair (5′ → 3′) |
|---|---|
| Fc (for construction of Fc alone and Fc-glucagon) | Forward, 5′-AAGCTTGGCATGGATCAATTC |
| Reverse, 5′-GGATCCAGGCCTACCCGCAGA | |
| Fc stop mutation | Forward, 5′-GACCATCTCCCGGTCTCCGGGTTAGCCTGGATCC |
| Reverse, 5′-GGACTAGTGGATCCAGGCTAACCCGGAGACCGGGAG | |
| Fc-glucagon frame shift | Forward, 5′-CCTGGATCCACTCAGTCCAGTGTGGTGG |
| Reverse, 5′-CCACCACACTGGACTGAGTGGATCCAGG | |
| Glucagona | Forward, 5′-GAATTCCATTCACAGGGAACA |
| Reverse, 5′-GCGGCCGCCTAGGTGTTCATCAG | |
| L26P-glucagon amplification | Forward, 5′-GAATTCCATTCACAGGGAACA |
| Reverse, 5′-GCGGCCGCCTAGGTGTTCATCGG | |
| R18Q-glucagon mutagenesis | Forward, 5′-AAATACCTGGACTCCCGCCAAGCCCAAGATTTTG |
| Reverse, 5′-CAAAATCTTGGGCTTGGCGGGAGTCCAGGTATTT | |
| L14P-glucagon mutagenesis | Forward, 5′-TACAGCAAATACCCGGGACTCCCGCCGAGCC |
| Reverse, 5′-GGCTCGGCGGGAGTCCGGGTATTTGCTGTA | |
| Fc (for construction with GLP-1, GLP-2, glicentin, MPGF) | Forward, 5′-GCTAGCATGGATCAATTCCGATGG |
| Reverse, 5′-AAGCTTACCCGGAGACCGGGAGATGG | |
| GLP-1(1–37)b | Forward, 5′-GGATCCCACGATGAGTTTGAGAGG |
| Reverse, 5′-GAATTCTCCTCTGCCTTTCACC | |
| GLP-1(7–37)b | Forward, 5′-GGATCCCACGCTGAAGGGACC |
| Reverse, 5′-GAATTCTCCTCTGCCTTTCACC | |
| GLP1 Glu-27 mutagenesis | Forward, 5′-GGCCAGGCTGCAAAGGAGTTCATTGCTTGG |
| Reverse, 5′-CCAAGCAATGAACTCCTTTGCAGCCTGGCC | |
| GLP-1 stop codon mutagenesis | Forward, 5′-GGTGAAAGGCAGAGGATGAGAATTCTGCA GATATCCTTAAG |
| Reverse, 5′-AAGGATATCTGCAGAATTCTCATCCTCTGCC TTTCACCAGC | |
| GLP-2b | Forward, 5′-GGATCCCATGCGGACGGCTCCTTC |
| Reverse, 5′-GAATTCGTCAGTGATTTTGGTTTG | |
| GLP-2 stop codon mutagenesis | Forward, 5′-CAAACCAAAATCACTGACTAGGAATTCTGCAGA TATCCTTAAGT |
| Reverse, 5′-AGGATATCTGCAGAATTCCTAGTCAGTGATTTT GGTTTGAATC | |
| D3Q-GLP-2 mutagenesis | Forward, 5′-GGATCCCATGCGCAGGGCTCCTTCTCC |
| Reverse, 5′-GGAGAAGGAGCCCTGCGCATGGGATCC | |
| D8K,E9K,N11K,D15K GLP-2 mutagenesis | Forward, 5′-GCTCCTTCTCCAAGAAGATGAAGACGATTCTCAAGAGTCTTGCC |
| Reverse, 5′-GGCAAGACTCTTGAGAATCGTCTTCATCTTCTTGGA GAAGGAGC | |
| Glicentinb | Forward, 5′-GGATCCCATTCCCTTCAGGACACGGAGG |
| Reverse, 5′-GAATTCCTAGCGTTTGGCAATGTTGTTCCTGTTC | |
| MPGFb | Forward, 5′-GGATCCCACGATGAGTTTGAGAGGCACGC |
| Reverse, 5′-GAATTCCTATTTCTTGTCAGTGATTTTGGTTTGAATCA | |
| Oxyntomodulinb | Forward, 5′-CTCGGATCCCATTCACAGGGAACATTCACCAGTGACTACAG |
| Reverse, 5′-GTGAATGGGATCCGAGCTCGGTACCAAGCTTACCCG |
a Constructs used a flexible 10-amino acid linker (sequence GSTQSSVVEF).
b Constructs used a flexible 8-amino acid linker (sequence KLGTELGS).