Gene cloning and production of active recombinant Brugia malayi microfilarial chitinase.
Canlas and coworkers [Canlas et al. (1984) Am. J. Trop. Med. Hyg. 33, 420-424] isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds. The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1548-1552). This paper describes the production of enzymatically active Bm Cht in Escherichia coli. Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold. The specific activity of the soluble MBP::Cht isolated from the E. coli cytoplasm was low. Exporting MBP::Cht into the E. coli periplasmic space increased the specific activity by 12-fold. This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.
Southworth
M W
MW
New England Biolabs, Inc., Beverly, MA 01915, USA.
Fuhrman
J A
JA
Robbins
P W
PW
Beauregard
K
K
Perler
F B
FB
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008842:Microfilariae / Q000201:enzymology / Q000235:genetics; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D002688:Chitinases