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. Author manuscript; available in PMC: 2017 Oct 31.
Published in final edited form as: Protein Expr Purif. 2012 Dec 13;88(1):98–106. doi: 10.1016/j.pep.2012.11.018

Table 1.

Purification of Recombinant SaPyrC

Step Volume (ml) Concentration (mg/ml) d, e Total Protein (mg)e Total Enzyme Activity (Units)f, g Specific Activity (Units/mg) h Yield (%)i Purity (%) i
Crude Lysatea 60 7.7 462 (354) 61.8 (72.7) 0.21 100 29
Clarified Extract 57 7.5 427 (327) 60.9 (71.6) 0.22 98 31
Tagged SaPyrC HisTrapb 17.6 6.6 116.2 (89) 37.6 (44.2) 0.50 61 70
Native SaPyrC HisTrapc 18.4 3.7 68.1 39.3 0.58 54 81
Native SaPyrC size exclusion (pooled peak) 17.0 2.5 42.5 28.7 0.68 39 95
a

From 5.77 g of wet weight E. coli cell pellet (from 1 L culture).

b

First Nickel HisTrap Column FPLC purification step (pooled fractions).

c

Second Nickel HisTrap Column FPLC purification step, after tag cleavage (pooled fractions).

d

The protein concentration was determined by Bradford assay using BSA as the standard.

e

Parenthetical values reflect calculated mass of native protein.

f

Enzyme activity measured by UV assays as described in methods (200 μL volume, 2 μL protein, 0.44 cm pathlength; 1.17 mM−1 cm−1 extinction coefficient).

g

Parenthetical values reflect estimated activity based upon lower enzyme efficiency of tagged SaPyrC (see Discussion).

h

Specific Activity calculated from parenthetical estimates of Total Protein and Total Activity for tagged protein.

i

Yield is calculated from Total Enzyme Activity; Purity is calculated from Specific Activity, with 95% final purity estimated the gel.