TABLE 1.
Construction and mutagenesis primers
| Primer Set | Mutant | Template | Primer sequence |
|---|---|---|---|
| 1 | NRP-2 KpnI insertion after Cys592 | NRP-2 | 5′-GAGGTGCTGGGCTGTGGTACCATGGACTGGACAG-ACTCC-3′ |
| 5′-GGAGTCTGTCCAGTCCATGGTACCACAGCCCAGC-ACCTC-3′ | |||
| 2 | NRP-2 signal sequence amplification including HindIII and KpnI sites | NRP-2 | 5′-GTTAAGCTTATGGATATGTTTCCTCTCACCTGGG-3′ |
| 5′-AATGGTACCGCCTCTCACTTGGTGTCTTGA-3′ | |||
| 3 | MAM domain amplification with KpnI and XbaI sites | NRP-2 | 5′-CGGGGTACCTCGGGATTCAATTGCAACTTC-3′ |
| 5′-CCTTCTAGAGCTGCCTCGGAGCAGCACTTT-3′ | |||
| 4 | NRP-2 EcoRV insertion between Pro641 and Ser642 | NRP-2 | 5′-GCAGCTCCCTGATATCTCGGGATTCAATTG-3′ |
| 5′-CGTCGAGGGACTATAGAGCCCTAAGTTAAC-3′ | |||
| 5 | Nrp-2 NheI insertion between Glu802 and Pro803 | NRP-2 | 5′-GAACTGCATGGAAGCTAGCCCCATCTCGGC-3′ |
| 5′-CTTGACGTACCTTCGATCGGGGTAGAGCCG-3′ | |||
| 6 | Nrp-1 EcoRV insertion between Pro644 and Thr645 | NRP-1 | 5′-CAATCAGAGTTTCCAGATATCACATATGGTTTT-AAC-3′ |
| 5′-GTTAGTCTCAAAGGTCTATAGTGTATACCAAAA-TTG-3′ | |||
| 7 | Nrp-1 NheI insertion between Lys811 and Pro812 | NRP-1 | 5′-GAAGATTGTGCAAAAGCTAGCCCAGCAGACCTG-3′ |
| 5′-CTTCTAACACGTTTTCGATCGGGTCGTCTGGAC-3′ | |||
| 8 | NRP-1Δ2 remove EcoRV | NRP-1Δ2 + restriction sites | 5′-CAATCAGAGTTTCCATCGGGATTCAATTG-3′ |
| 5′-GTTAGTCTCAAAGGTAGCCCTAAGTTAAC-3′ | |||
| 9 | NRP-1Δ2 remove NheI | NRP-1Δ2 + restriction sites | 5′-GAACTGCATGGAACCAGCAGACCTG-3′ |
| 5′-CTTGACGTACCTTGGTCGTCTGGAC-3′ | |||
| 10 | NRP-2Δ1 remove EcoRV | NRP-2Δ1 + restriction sites | 5′-GCAGCTCCCTACATATGGTTTTAAC-3′ |
| 5′-CGTCGAGGGATGTATACCAAAATTG-3′ | |||
| 11 | NRP-2Δ1 remove NheI | NRP-2Δ1 + restriction sites | 5′--GAAGATTGTGCAAAACCCATCTGCGC--3′ |
| 5′-CTTCTAACACGTTTTGGGTAGACGCG-3′ | |||
| 12 | NRP-2Δ1 (to remove stop codon inserted during MAM swap at Glu622) | NRP-2Δ1 | 5′-CCACCGAAGAGGAGGCCACAGAGTG-3′ |
| 5′-GGTGGCTTCTCCTCCGGTGTCTCAC-3′ | |||
| 13 | Amplification of ectodomains to make NRP-Fc constructs | NRP-1 and NRP-1Δ2 | 5′-AGACCCAAGCTGGCCATGGAGAGGGGGCTGCCGCTCCTC-3′ |
| 5′-AGACACCCTCCCTCCGGGGTCTAAGGTCTTCAACACATT-3′ | |||
| 14 | Amplification of ectodomains to make NRP-Fc constructs | NRP-2Δ1 | 5′-GAGACCCAAGCTGGCCATGGATATGTTTCCTCTCACCTGG-3′ |
| 5′-AGACACCCTCCCTCCGGGATCCAGGGTGTACAGCCAGCTC-3′ | |||
| 15 | Amplification of pcDNA4-hFc vector for In-Fusion | pcDNA4-NRP-2-hFc | 5′-GCTCTAGAGGGCCCGCGGTTCGAAGGTA-3′ |
| 5′-AGTCTAGAGGGCCCGCGGTTC-3′ | |||
| 16 | Insertion of splice consensus sequence | NRP-2-Fc and NRP-2Δ1-Fc | 5′-CACCCTGGATCCCACAGGTAAGTGGAGGGAG-GGTGT-3′ |
| 5′-ACACCCTCCCTCCACTTACCTGTGGGATCCAGGGTG-3′ | |||
| 17 | Insertion of splice consensus sequence | NRP-1-Fc and NRP-1Δ2-Fc | 5′-AGACCTTAGACCCCACAGGTAAGTGGAGGGAGGGTGTC-3′ |
| 5′-GACACCCTCCCTCCACTTACCTGTGGGGTCTAAGGTCT-3′ | |||
| 18 | Amplification of NRP-1 linker region through the cytoplasmic tail for NRP-2ΔLM1 construct | NRP-1 | 5′-GAGGTGCTGGGCTGTGAAGTGGAAGCCCCTACAGCTGGA-3′ |
| 5′-CGGGCCCTCTAGAGCTGCCTCCGAATAAGTACTCTGTG-3′ | |||
| 19 | Amplification of NRP-2 linker region through the cytoplasmic tail for NRP-1ΔLM2 construct | NRP-2 | 5′-GAGCTGCTGGGCTGTGACTGGACAGACTCCAAGCCCACG-3′ |
| 5′-CGGGCCCTCTAGAGCTGCCTCGGAGCAGCACTTTTGGT-3′ | |||
| 20 | Amplification of NRP-2 sequences except the linker region through the cytoplasmic tail sequences in pcDNA3.1/V5-His B vector | NRP-2 | 5′-ACAGCCCAGCACCTCCAGCCGCATCCCA-3′ |
| 5′-GCTCTAGAGGGCCCGCGGTTCGAAGGTA-3′ | |||
| 21 | Amplification of NRP-1 sequences except the linker region through the cytoplasmic tail in pcDNA3.1/V5-His B vector | NRP-1 | 5′-ACAGCCCAGCAGCTCCATTCTGAGCCCCA-3′ |
| 5′-GCTCTAGAGGGCCCGCGGTTCGAAGGTA-3′ | |||
| 22 | Deletion of extra cytosine base | NRP-1ΔLM2 | 5′-GACAACCACCCCCTACCCCACCGAAG-3′ |
| 5′-CTTCGGTGGGGTAGGGGGTGGTTGTC-3′ | |||
| 23 | Amplification of ectodomains to make NRP-Fc constructs | NRP-2ΔLM1 | 5′-GAGACCCAAGCTGGCCATGGATATGTTTCCTCT-CACCTGG-3′ |
| 5′-GACACCCTCCCTCCACTTACCTGTGGGGTCTAAGGTCT-3′ | |||
| 24 | Amplification of ectodomains to make NRP-Fc constructs | NRP-1ΔLM2 | 5′-AGACCCAAGCTGGCCATGGAGAGGGGGCTGCCGCTCCTC-3′ |
| 5′-ACACCCTCCCTCCACTTACCTGTGGGATCCAGGGTG-3′ | |||
| 25 | Inverse PCR of NRP-2-Fc to eliminate MAM domain | NRP-2-Fc | 5′-CCTCCACTTACCTGTAGGGAGCTGCAAATCTTTGTCATCCTCA-3′ |
| 5′-GGAGGGAGGGTGTCTGCTGGAA-3′ | |||
| 26 | NRP-2 E652A | NRP-2-Fc | 5′-CTTCGATTTCCTCGCGGAGCCCTGTGG-3′ |
| 5′-GAAGCTAAAGGAGCGCCTCGGGACACC-3′ | |||
| 27 | NRP-2 E653A | NRP-2-Fc | 5′-CGATTTCCTCGAGGCGCCCTGTGGTTGG-3′ |
| 5′-GCTAAAGGAGCTCCGCGGGACACCAACC-3′ | |||
| 28 | NRP-2 E652A/E653A | NRP-2-Fc | 5′-CTTCGATTTCGCGGCGCCCTGTGGTTGG-3′ |
| 5′-GAAGCTAAAGCGCCGCGGGACACCAACC-3′ | |||
| 29 | NRP-2 D683A | NRP-2-Fc | 5′-GACGTTTCCAGATGCCAGGAATTTCTTGCG-3′ |
| 5′-CTGCAAAGGTCTACGGTCCTTAAAGAACGC-3′ |