Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland.
The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities.
Roberto
Patrícia G
PG
Unidade de Biotecnologia, Universidade de Ribeirão Preto, UNAERP, Ribeirão Preto, SP, Brazil.
Kashima
Simone
S
Soares
Andreimar M
AM
Chioato
Lucimara
L
Faça
Victor M
VM
Fuly
André L
AL
Astolfi-Filho
Spartaco
S
Pereira
José O
JO
França
Suzelei C
SC
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D017837:Bothrops / Q000033:anatomy & histology / Q000378:metabolism; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D054501:Group II Phospholipases A2; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D030162:Reptilian Proteins; D014757:Viper Venoms / Q000737:chemistry / Q000201:enzymology
D003435:Crotalid Venoms; D004792:Enzyme Precursors; D010975:Platelet Aggregation Inhibitors; D030162:Reptilian Proteins; D014757:Viper Venoms; D010741:Phospholipases A; C495705:BOJU-III protein, Bothrops jararacussu; D054501:Group II Phospholipases A2; D054467:Phospholipases A2