title: Development of a bacterial cloning vector for expression of scorpion toxins for biotechnological studies.
abstract: 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.
authors: Department of Microbiology and Immunology, Biology Institute, Campinas University, Campinas, SP, Brazil. eliana.stehling@gmail.com
authors: Eliana Guedes
authors: EG
authors: Stehling
authors: Wanderley Dias
authors: WD
authors: da Silveira
authors: Tatiana Amabile
authors: TA
authors: Campos
authors: Marcelo
authors: M
authors: Brocchi
authors: Thelma Aguiar
authors: TA
authors: Pertinhez
authors: Alberto
authors: A
authors: Spisni
mesh_terms: 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
chemical_list: D000939:Epitopes; D009841:Oligonucleotides; D012604:Scorpion Venoms; D014760:Viral Fusion Proteins; D006639:Histidine; D010450:Endopeptidases