Biomedical Engineering Reference
In-Depth Information
incorporation and expression of the ANIBSEC or ANICSEC-2 vector by
assaying extracellular media for α-1,2-mannosidase activity.
Protein Expression
Fresh liquid cultures were prepared for protein expression by inoculation
of 50 ml YFT media with 200 µl of conidial suspensions. These cultures
were grown on a rotary shaker (200 rpm) at 30°C for 24-72 hours. The YFT
media contains limited amounts of glucose that represses the inducible
alcA
promoter. After 36-40 hours, the glucose is depleted and the cultures
shift to fructose as a carbon source. This releases the glucose repression
of the
alcA
promoter and allows expression of
alcA
-driven genes. After
65 hours, mycelium was removed by fi ltration through cheesecloth
followed by centrifugation. Cleared supernatants were used for screening
transformants for mannosidase activity and for further purifi cation steps.
Mannosidase Assays
Mannosidase assays were performed using the disaccharide Man-α-
1,2-Man-OCH
3
as a substrate in a coupled enzyme assay as described
earlier (Scaman et al. 1996), with some modifi cations. Digestion of the
substrate was performed in a 30 µl fi nal volume containing e in 10 mM
sodium acetate/acetic acid buffer (pH5.0) and 3 µl 100 mM disaccharide
Man-α-1,2-Man-OCH
3
incubated at 37°C for 3 hours. Released mannose
was detected by addition of 30 µl 1.25M Tris-HCl (pH7.6) and 240 µl of
developing solution, containing glucose oxidase (55 U/ml), horseradish
peroxidase (1 U/ml) and o-dianisidine dihydrochloride (70 µg/ml)
followed by incubation at 37°C for 3 hours. Absorbance measurements
at 450 nm were used to determine fi nal color change. A standard curve of
free mannose concentrations was determined for comparison. One unit of
enzyme activity was defi ned as the amount of enzyme that released 1 nmol
of mannose per hour. Specifi c α-mannosidase activity was standardized by
comparison with total protein in the enzyme extracts and was defi ned as
the units of enzyme activity per µg of total protein. Protein concentrations
were determined by the Bradford method (Bradford 1976) and comparison
to a bovine serum albumin (BSA) standard curve.
Enzyme Purifi cation
Proteins were precipitated from cleared supernatants by stepwise addition
of ammonium sulfate followed by centrifugation after each addition.
Mannosidase activity precipitated mainly in the 40-70% ammonium
