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column of Superdex 200 HR 10/30, previously calibrated with protein molecular mass
standards, was 195,550 Da. Hence, it is assumed that the purified Arthrobacter sp. 32c
β-D-galactosidase is probably a trimeric protein.
Table 1. Purification of recombinant Arthrobacter sp. 32c
β
-D-galactosidase.
Purifi cation step
Volume
(ml)
Protein (mg)
Specifi c activity
(U mg- 1 )
Total activity
(U)
Purifi cation
(fold)
Recovery
(%)
E coli LMG plysN pBADMyc-HisA-32cβ-gal
Cell extract
30
580
13.8
8004
1.0
100
Affi nity chromatography
3.2
27
155.9
4209
21.0
53
P. pastoris GS 11S pPICZaA-32cβ-gal
Broth
1000
3400
28.7
97580
1.0
100
Protein precipitation
54
340
136.1
46274
10.0
47
Affi nity chromatography
11
137
154.7
21194
24.8
22
P. pastoris GS 11S pGAPZaA-32cβ-gal
Broth
1000
5200
16.2
84240
1.0
100
Protein precipitation
46
450
102.7
46215
11.6
55
Affi nity chromatography
10
97
153.1
14851
53.6
18
Figure 2. SDS-PAGE analysis of the expression and purification steps of the Arthrobacter sp. 32c
-D-
β
galactosidase expressed by E. coli host (A), P. pastoris GS115 pPICZ
A-32c
-gal methanol induced
α
β
variant (B) and P. pastoris GS115 pGAPZ
-gal constitutive variant (C). Lanes 1 protein weight
marker. Panel A: lane 2cell extract after expression, lane 3purified
A-32c
α
β
-D-galactosidase after affinity
chromatography. Panel B and C: lane 2broth after protein expression, lane 3protein precipitate, lane
4purified
β
-D-galactosidase after affinity chromatography.
β
 
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