TABLE 2.
Oligonucleotides used to introduce truncations into the C terminus of the C370 sequence and amino acid substitutions in the LPQTG motifa
| Mutation | Nucleotide exchange | Primer pair |
|---|---|---|
| 249P(A) | 1868Proline→alanine | 5′-CATAAGCAAACTCTATTGGCTCAAACTGGTACTGAAAC3′ |
| 5′-GTTTCAGTACCAGTTTGAGCCAATAGAGTTTGCTTATG-3′ | ||
| 249P(N) | 1868Proline→asparagine | 5′-CATAAGCAAACTCTATTGAATCAAACTGGTACTGAAAC3′ |
| 5′-GTTTCAGTACCAGTTTGATTCAATAGAGTTTGCTTATG-3′ | ||
| 249T(A) | 1870Threonine→alanine | 5′-CAAACTCTATTGCCTCAAAGTGGTACTGAAACTAA-3′ |
| 5′-GTTAGTTTCAGTACCAGTTTGAGGCAATAGAGTTTG-3′ | ||
| 249T(G) | 1870Threonine→glycine | 5′-CAAACTCTATTGCCTCAAGGTGGTACTGAAACTAAC-3′ |
| 5′-GTTAGTTTCAGTACCACCTTGAGGCAATAGAGTTTG-3′ | ||
| 249T(S) | 1870Threonine→serine | 5′-CAAACTCTATTGCCTCAAAGTGGTACTGAAACT-3′ |
| 5′-GTTAGTTTCAGTACCACTTTGAGGCAATAGAGTTTG-3′ | ||
| 249G(A) | 1871Glycine→alanine | 5′-CTCTATTGCCTCAAACTGCTACTGAAACTAACCCAC-3′ |
| 5′-GTGGGTTAGTTTCAGTAGCAGTTTGAGGCAATAGAG-3′ |
a
The C-terminal truncated forms were generated by PCR using primers 5′-GCGCTCTAGATTAAAAAATTCCTGCGCCTAATG-3′ (truncation 249-10), (5′-GCGCTCTAGATTATGCAAAAATTCCTGCGCCTAATG-3′) (truncation 249-9), and 5′-GCGCTCTAGATTACTTTGCAAAAATTCCTGCGCC-3′ (truncation 249-8) (restriction sites introduced for subcloning are underlined). Single-amino-acid exchanges in the LPQTG motif were introduced by site-directed mutagenesis by a PCR approach using the primers given. Underlined nucleotides indicate those amino acids that are substituted by site-directed mutagenesis.