Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells.
Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.
Cadel
Sandrine
S
Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, FRE 2621, Université Pierre et Marie Curie--Centre National de la Recherche Scientifique, 96 Boulevard Raspail, 75006 Paris, France.
Gouzy-Darmon
Cécile
C
Petres
Stéphane
S
Piesse
Christophe
C
Pham
Viet-Laï
VL
Beinfeld
Margery C
MC
Cohen
Paul
P
Foulon
Thierry
T
D000626:Aminopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007313:Insecta / Q000166:cytology / Q000821:virology; D008297:Male; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013737:Testis / Q000378:metabolism
D018076:DNA, Complementary; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000626:Aminopeptidases; C019721:aminopeptidase B