Abstract
The screening for the Aspergilus niger glucoamylase cDNA has led to the discovery that a full length cDNA in a pUC vector is lethal to Escherichia coli. Initial screening has indicated that full length glucoamylase cDNAs were initially present in the cDNA libraries but deletion mutants arose during the purification of the clones. The deletion mutants contained identical incomplete cDNAs lacking 35 nucleotides downstream of the initiation codon. The missing portion encodes part of an 18 amino acid signal peptide responsible for the translocation of the A. niger glucoamylase extracellularly. Therefore, to reconstruct a full length cDNA, synthetic oligonucleotides which represent the missing portion of the gene were ligated with the incomplete cDNA. However, several attempts to reconstruct a full length cDNA failed when the cDNA was in pUC18, a high copy number expression vector which utilizes the lac promoter. Fusion of a double stranded oligonucleotide representing the missing portion of the cDNA with a truncated cDNA led to the isolation of one deletion mutant which completely lost the entire glucoamylase cDNA except for the signal sequence. Incomplete cDNA lacking part of the signal sequence was then subcloned into a promoterless vector pSP72 and ligated with synthetic oligonucleotides representing the missing portion. This finally yielded full length 2.0 kb glucoamylase cDNA. In addition, it was found that subcloning of the full length glucoamylase cDNA into pT7T3 18U, another high copy number vector equipped with the lac promoter, also resulted in deletion of part of the cDNA to 1.5 kb.