MRNA secondary structure can greatly affect production of recombinant phospholipase A(2) toxins in bacteria

摘要

The neurotoxic activity of ammodytoxin A (AtxA), a phospholipase A, from Vipera ammodytes ammodytes venom, has been investigated by protein engineering. With the aim of obtaining AtxA as a non-fused protein in the bacterial cytoplasm and avoiding problems with incomplete cleavage in vivo of the initial Met preceding the first residue (Ser1), a double mutant (S1A/E4Q) was prepared and expressed in Escherichia coli. Immunoblotting of the bacterial lysate showed that the mutant was synthesized at a low level not exceeding 0.5% of total cell protein. Analysis of the potential secondary structure of the mutant mRNA in the translation initiation region suggested that the Ala I (GCC) and Leu2 (CUG) codons used are likely to be involved in a hairpin structure with the Thr13 (ACG) and Gly14 (GGG) codons. hindering effective translation at the ribosome. To weaken this structure (by DeltaG of about 20 kJ/mol) the same double mutant was prepared using another mutagenic oligonucleotide with silent mutations in the Ala1 (GCU) and Leu2 (UUG) codons. The mutant was successfully produced at a level of approximately 15% of total protein, with the initial Met completely removed in the bacterial cell. Such an approach could be important in solving similar problems in bacterial production of other toxic proteins.