Bacterial expression and characterization of functional recombinant triosephosphate isomerase from Schistosoma japonicum.
The dimeric enzyme triosephosphate isomerase (TPI) converts glyceraldehyde-3-phosphate to dehydroxyacetone phosphate, a key reaction in glycolysis. Previous studies of the native enzyme in the human blood-flukes belonging to the genus Schistosoma have indicated that TPI is a promising anti-schistosome vaccine antigen. However, a recombinant form of the enzyme is required as an alternative to the impractical option of using biochemically purified TPI obtained from worm tissue for large-scale vaccine use. We previously cloned and sequenced a full-length cDNA encoding the TPI of the Asian (Chinese strain) schistosome Schistosoma japonicum (SjcTPI). We now report very high level bacterial expression of this cDNA and the subsequent purification of the recombinant protein to >98% homogeneity under nondenaturing conditions. The recombinant SjcTPI (re-SjcTPI) was shown to be enzymatically active with a specific activity of 7687 units/mg protein, an activity higher than that of commercially obtained porcine TPI tested concurrently under the same assay conditions. The K(m) value for the re-SjcTPI using glyceraldehyde-3-phosphate as substrate was 406.7 microM, which is similar to the K(m) values reported for the yeast enzyme and various mammalian TPIs. With the availability of substantial amounts of enzymatically active and readily purified re-SjcTPI made in bacteria we can now test whether the recombinant protein can induce a similar level of protection in vaccination/challenge experiments as the native, biochemically purified enzyme.
Sun
W
W
Australian Centre for International and Tropical Health and Nutrition, The University of Queensland and The Queensland Institute of Medical Research, 300 Herston Road, Queensland, 4029, Australia.
Liu
S
S
Brindley
P J
PJ
McManus
D P
DP
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012549:Schistosoma japonicum / Q000201:enzymology*; D013053:Spectrophotometry; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D014305:Triose-Phosphate Isomerase