mRNA secondary structure can greatly affect production of recombinant phospholipase A(2) toxins in bacteria.
The neurotoxic activity of ammodytoxin A (AtxA), a phospholipase A(2) 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 Ala1 (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.
Ivanovski
G
G
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000, Ljubljana, Slovenia.
Gubensek
F
F
Pungercar
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014757:Viper Venoms / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014757:Viper Venoms; C068117:ammodytoxin A; D010741:Phospholipases A