Dg. Moran et al., CHARACTERIZATION OF RECOMBINANT HUMAN MAN(9)-MANNOSIDASE EXPRESSED INESCHERICHIA-COLI, Journal of fermentation and bioengineering, 86(3), 1998, pp. 277-283
Man(9)-mannosidase is an alpha 1,2-specific exo-enzyme involved in the
N-linked oligosaccharide processing pathway, In this study, the expre
ssion of the truncated gene encoding for the human Man(9)-mannosidase
(amino acids 140 to 625) in Escherichia coli facilitated further chara
cterization of the enzyme. PCR primers were designed to isolate a trun
cated human kidney Man(9)-mannosidase gene, The gene was fused to a T7
protein tag and six histidine residues at the N and C-terminal ends,
respectively. The truncated Man(9)-mannosidase enzyme was purified by
affinity chromatography and ion exchange chromatography. Activity of t
he purified enzyme was examined by HPLC and pyridylaminated (PA) oligo
saccharides as substrates. Results showed that Man(9) GlcNAc(2) is rap
idly converted to Man(6)GlcNAc(2). Substrate specificity was analyzed
using different isomers of Man(6)GlcNAc(2), Man(7)GlcNAc(2) and Man(8)
GlcNAc(2), The truncated Man(9)-mannosidase exhibited very little acti
vity towards the alpha 1,2-linked terminal mannose residue on the alph
a 3 branch of the Man(9)GlcNAc(2), a characteristic of the pig and cal
f liver Man(9)-mannosidases. Based on the HPLC analysis of the reactio
n products using size-fractionation and reversed-phase columns, the pa
thway for Man(9)GlcNAc(2) trimming was deduced. The recombinant enzyme
had optimum activity at 37-40 degrees C and pH 6.5-7.0. Enzyme activi
ty was inhibited by 100 mu M deoxymannojirimycin (dMNJ), Expression of
the human kidney Man(9)-mannosidase gene in E. coli Facilitated the d
etailed characterization of the enzyme which until now had not been ex
pressed in amounts necessary for biochemical and physical analyses.