Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica)

Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inc...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2012-10, Vol.427 (2), p.321-325
Main Authors: Yunes Quartino, Pablo Javier, Barra, José Luis, Fidelio, Gerardo Daniel
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Bothrops - genetics
Bothrops - metabolism
Cloning, Molecular
Hydrolysis
Molecular Sequence Data
Phospholipases A2, Secretory - chemistry
Phospholipases A2, Secretory - genetics
Protein Conformation
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
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Summary:Bothrops diporus is a very common viper in Argentina. At present, no complete sequence of secreted phospholipase A(2) (sPLA(2)) from this snake has been reported. We have cloned two sPLA(2) isoenzymes as well as a putative sPLA(2)-like myotoxin from venom gland. The two sPLA(2) were expressed as inclusion bodies in Escherichia coli with an N-terminal tag of ubiquitin. After in vitro renaturation and cleavage step, using an ubiquitin specific peptidase, the recombinants exhibited sPLA(2) activity when analyzed by means of Langmuir dilauroylphosphatidylcholine monolayers as substrate. Both enzymes have a similar surface pressure-activity profile when compared with non-recombinant purified isoforms. To our knowledge, this is the first time that analysis of optimal lateral pressure of substrate monolayers by using the surface barostat technique is performed on recombinant sPLA(2)s.
ISSN:1090-2104
DOI:10.1016/j.bbrc.2012.09.051