Development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies.
Scorpion venoms contain toxic peptides that recognize K(+) channels of excitable and non-excitable cells. These toxins comprise three structurally distinct groups designated alpha-KTx, beta-KTx, and gamma-KTx. It is highly desirable to develop systems for the expression of these toxins for further physiological and structural studies. In this work, an expression vector (pTEV3) was constructed by inserting protein D (major capsid of phage lambda) and TEV protease recognition site into plasmid pET21d DNA sequences. Three alpha-KTx toxins (OsK2, PbTx1, and BmKK3) were cloned into vector pTEV3 and expressed as soluble fusion proteins. The fractions containing the purified fusion proteins (protein D-toxin) were treated with TEV protease to remove protein D. The resulting toxins were analyzed by MALDI-TOF Mass Spectrometry. The results showed that the vector is appropriate for the expression of the target toxins in soluble form and that ion exchange purification of these toxins by flow-through recovery is possible. Analysis by MALDI-TOF Mass Spectrometry of Osk2 demonstrated that this toxin was expressed in its native form, as suggested by the values expected for the presence of two disulfide bridges.
Stehling
Eliana Guedes
EG
Department of Microbiology and Immunology, Biology Institute, Campinas University, Campinas, SP, Brazil. eliana.stehling@gmail.com
da Silveira
Wanderley Dias
WD
Campos
Tatiana Amabile
TA
Brocchi
Marcelo
M
Pertinhez
Thelma Aguiar
TA
Spisni
Alberto
A
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics*; D010582:Bacteriophage lambda / Q000737:chemistry; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D002213:Capsid / Q000737:chemistry; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000494:pharmacology; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000096:biosynthesis; D010957:Plasmids; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012605:Scorpions / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014169:Transformation, Bacterial; D014760:Viral Fusion Proteins / Q000378:metabolism
D000939:Epitopes; D009841:Oligonucleotides; D012604:Scorpion Venoms; D014760:Viral Fusion Proteins; D006639:Histidine; D010450:Endopeptidases