Synthesis and expression of the gene coding for noxiustoxin, a K+ channel-blocking peptide from the venom of the scorpion Centruroides noxius.
A set of six synthetic overlapping oligonucleotides coding for noxiustoxin were coupled into a continuous DNA fragment by means of recursive polymerase chain reaction. The polymerase chain reaction product was digested with SalI and HindIII, ligated into the E, coli vector pCSP 105 and expressed as a fusion protein. The fusion protein was purified and digested with trypsin and the hydrolysis products were separated by high-performance liquid chromatography. Approximately 1.3 mg of recombinant noxiustoxin per liter of culture was obtained. Amino acid analysis and N-terminal amino acid sequence of the recombinant noxiustoxin confirmed the nucleotide sequence of the cloned DNA. Binding experiments using rat brain synaptosomal membranes revealed that recombinant noxiustoxin displaced bound radioactive native NTX with a similar efficiency to cold native noxiustoxin.
Martinez
F
F
Department of Molecular Recognition and Structural Biology, Biotechnology Institute, National Autonomous University of Mexico.
Becerril
B
B
Gurrola
G B
GB
Martin
B M
BM
Possani
L D
LD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain; D002851:Chromatography, High Pressure Liquid; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D026902:Potassium Channel Blockers; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D012605:Scorpions; D013574:Synaptosomes / Q000378:metabolism
D010455:Peptides; D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C052567:noxiustoxin