Construction of a cDNA Encoding a Repetitive Amino Acid Sequence

PCR is a powerful method with which to generate almost any desired cDNA sequence. Construction of cDNA fragments that encode repetitive amino acid sequences by PCR has proven problematic, however, because repetitive nucleotide primers tend to amplify undesired fragments because of their mis-annealing with the template. To overcome this problem, Dombrowski and Wright (2) developed a solid-phase gene assembly protocol, but this technique requires preparation of a solid-phase reaction system and thus cannot be routinely performed in most laboratories. Here, we describe a new strategy that makes use of standard PCR protocols, yet enables assembly of cDNAs encoding repetitive amino acid sequences. The peptide unit Ala-Thr-Pro-AlaPro (ATPAP) is reported to be the shortest that can still function as an acceptor for N-acetylgalactosaminyltransferase-I (4). By introducing this peptide into a secretable form of fibroblast growth factor (FGF) (3) and expressing it in Chinese hamster ovary cells, we were able to produce FGF modified with a single O-glycan moiety (1). Subsequently, neo-O-glycosylated forms of FGF containing multiple O-glycans, similar to mucin-type glycoproteins, were created using two cDNAs encoding FGFs with 10 repeats of the pentapeptide O-glycosylation unit at the Nor C-terminus. In this report, the creation of the N-terminal modified form is described in detail. The amino acids comprising the pentapeptide O-glycosylation unit, alanine, proline and threonine, are encoded by codons GCX, CCX and ACX, respectively, where X is any nucleotide. We therefore designed four nucleotide cassettes that each encoded ATPAP and differed from one another at the last nucleotide for each codon (Figure 1A). To avoid mis-annealing the cassettes, they were linked together so that they encoded more than three repeats of the ATPAP unit (Figure 1B). Generation of clones encoding the modified FGF entailed performing two rounds of PCR. The primers for the first PCR were 5′-TTCGAATTCCCAAGGCACGCTACTGGATGCTACTCCTGCTCCTGCGACGCCGGCGCCGGCA-3′ Benchmarks