Giuliani C D, Iemma M R, Bondioli A C, Souza D H, Ferreira L L, Amaral A C, Salvini T F, Selistre-de-Araujo H S
Departamento de Ciências Fisiológicas, Universidade Federal de São Carlos, 13565 São Carlos, SP, Brazil.
Toxicon. 2001 Oct;39(10):1595-600. doi: 10.1016/s0041-0101(01)00142-8.
ACL myotoxin (ACLMT) is a K49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus (broad-banded copperhead) that induces necrosis of skeletal muscle. We have previously cloned and sequenced the cDNA coding for ACLMT from a venom gland cDNA library. In order to perform structure and function studies, we have developed an expression system for production of ACLMT as a fusion protein with maltose binding protein (MBP) from the periplasm of bacteria, using the pMAL-p2 expression vector. The cDNA coding for the mature toxin without the signal peptide was amplified by PCR and subcloned into the pMAL-p2 vector. The new plasmid (pMAL-MT) was used to transform BL21(DE3) E. coli cells. Culture of transformed cells induced with IPTG led to the expression of a 60 kDa fusion protein which strongly reacts with anti-native ACLMT antibodies. The fusion protein was purified from the bacterial periplasm by affinity chromatography in an amylose column and by gel filtration. The purified fusion protein (MBP-rACLMT) was able to induce necrosis of skeletal muscle of mice very similar to that caused by the native myotoxin.
蝮蛇肌毒素（ACLMT）是一种从蝮蛇（宽带铜头蝮）毒液中分离出的K49磷脂酶A2样蛋白，可诱导骨骼肌坏死。我们之前已从毒液腺cDNA文库中克隆并测序了编码ACLMT的cDNA。为了进行结构和功能研究，我们利用pMAL-p2表达载体开发了一种表达系统，用于从细菌周质中生产与麦芽糖结合蛋白（MBP）融合的ACLMT。通过PCR扩增编码无信号肽的成熟毒素的cDNA，并将其亚克隆到pMAL-p2载体中。用新质粒（pMAL-MT）转化BL21(DE3)大肠杆菌细胞。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导转化细胞培养，导致表达一种60 kDa的融合蛋白，该蛋白与抗天然ACLMT抗体强烈反应。通过在直链淀粉柱上的亲和层析和凝胶过滤从细菌周质中纯化融合蛋白。纯化的融合蛋白（MBP-rACLMT）能够诱导小鼠骨骼肌坏死，与天然肌毒素引起的坏死非常相似。
