Expression of an active recombinant lysine 49 phospholipase A2 myotoxin as a fusion protein in bacteria

被引:17
作者
Giuliani, CD
Iemma, MRC
Bondioli, ACV
Souza, DHF
Ferreira, LL
Amaral, AC
Salvini, TF
Selistre-de-Araujo, HS [1 ]
机构
[1] Univ Fed Sao Carlos, Dept Fisioterapia, BR-13565 Sao Carlos, SP, Brazil
[2] Univ Fed Sao Carlos, Dept Ciencias Fisiol, BR-13565 Sao Carlos, SP, Brazil
基金
巴西圣保罗研究基金会;
关键词
snake venom; phospholipase A(2); expression; refolding; myotoxin;
D O I
10.1016/S0041-0101(01)00142-8
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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. (C) 2001 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:1595 / 1600
页数:6
相关论文
共 29 条
[1]   Sequence analysis of LYS49 phospholipase A(2) myotoxins: A highly conserved class of proteins [J].
DeAraujo, HSS ;
White, SP ;
Ownby, CL .
TOXICON, 1996, 34 (11-12) :1237-1242
[2]   CDNA cloning and sequence analysis of a lysine-49 phospholipase A(2) myotoxin from Agkistrodon contortrix laticinctus snake venom [J].
deAraujo, HSS ;
White, SP ;
Ownby, CL .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1996, 326 (01) :21-30
[3]   Phospholipase A2 in eicosanoid generation [J].
Dennis, EA .
AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, 2000, 161 (02) :S32-S35
[4]  
DENNIS EA, 1994, J BIOL CHEM, V269, P13057
[5]   HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI AND RAPID PURIFICATION OF ENZYMATICALLY ACTIVE HONEY-BEE VENOM PHOSPHOLIPASE-A2 [J].
DUDLER, T ;
CHEN, WQ ;
WANG, SS ;
SCHNEIDER, T ;
ANNAND, RR ;
DEMPCY, RO ;
CRAMERI, R ;
GMACHL, M ;
SUTER, M ;
GELB, MH .
BIOCHIMICA ET BIOPHYSICA ACTA, 1992, 1165 (02) :201-210
[6]  
Falconi M, 2000, J MOL RECOGNIT, V13, P14, DOI 10.1002/(SICI)1099-1352(200001/02)13:1<14::AID-JMR484>3.0.CO
[7]  
2-F
[8]  
FLETCHER JE, 1997, VENOM PHOSPHOLIPASE
[9]   MYOTOXIN-II FROM BOTHROPS-ASPER (TERCIOPELO) VENOM IS A LYSINE-49 PHOSPHOLIPASE-A2 [J].
FRANCIS, B ;
GUTIERREZ, JM ;
LOMONTE, B ;
KAISER, II .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1991, 284 (02) :352-359
[10]   Interfacial recognition by bee venom phospholipase A2:: Insights into nonelectrostatic molecular determinants by charge reversal mutagenesis [J].
Ghomashchi, F ;
Lin, Y ;
Hixon, MS ;
Yu, BZ ;
Annand, R ;
Jain, MK ;
Gelb, MH .
BIOCHEMISTRY, 1998, 37 (19) :6697-6710