Cellufine Phosphate | For Purification of DNA-Binding Proteins and T7 RNA Polymerase

Affinity Chromatography Media

Cellufine^TMPhosphate

For purification of nucleic acid-binding proteins such as T7 RNA polymerase, which is an enzyme for synthesizing mRNA drugs

Cellufine Phosphate is an affinity media designed for concentration, purification of proteins and, enzymes such as nucleic acid related proteins. Base of media is spherical and rigid cellulose functionalized with Phosphate esters. T7 RNA polymerase, which is a synthase of mRNA drugs, is a nucleic acid-binding protein. So it can be efficiently purified by cellufine Phosphate.

Ligand structure of Cellufine Phosphate — Figure1Partial Structure of Cellufine Phosphate

Characteristics
Support Matrix	Cellulose
Ligand	Phosphate ester
Ligand conc.	0.3 - 0.8meq/ml
Adsorption Capacity	≧ 20mg/ml-gel (lysozyme)

Purification example of T7 RNA polymerase

T7 RNA polymerase is a important RNA synthase used to synthesize mRNA from template DNA in in vitro transcription. In this study, high-purity T7 RNA polymerase was purified by a two-step chromatography step of first performing crude purification with Cellufine MAX DEAE and then performing affinity purification with Cellufine Phosphate.

Chromatogram of T7 RNA polymerase purification — Figure 2 Purification of T7 RNA polymerase with Cellufine Phosphate

After purifying with Cellufine MAX DEAE, elution faction is purified with Cellufine Phosphate. T7 RNA polymerase is accumulated in the EL2 fraction displayed in red.

Table 2　Recovery of T7 RNA polymerase in each fraction after column

Fraction	Enzyme activity (Unit/protein)	Enzyme recovery (%)	Protein recovery (%)
load sample	94043	100	100
Flowthrough fraction	2763	1.8	59.8
Elution fraction	267034	70.2	24.7

Table 1 shows the enzyme activity and protein recovery after column purification with Cellufine Phosphate (Figure 1). The activity of T7 RNA polymerase in the eluted fraction was as high as 70.2%. The amount of protein was reduced to 24.7%, indicating that contaminants were efficiently removed.

Figure 3　SDS-PAGE after purification of Cellufine Phosphate

1: Lysate, 2: Ammonium sulfate precipitation, 3: Elution fraction of Cellufine MAX DEAE, 4: Elution fraction of Cellufine Phosphate, 5: Control

The degree of purification was evaluated by SDS-PAGE using fractions obtained from each purification process of Cellufine MAX DEAE and Cellufine Phosphate. Each step of chromatography removes contaminants and at the stage of Cellufine Phosphate, the T7 RNA polymerase can be purified to near single band.

Pore size of Cellufine Phosphate

Cellfine Phosphate Pore size characteristics — Figure2Calibration carves of Cellufine Phosphate and a conventional Cellulose phosphate.

C.I.P. Stability test

Cellufine Phosphate is stable to cleaning in place (C.I.P) by alkali.

Repeated use test data of Cellufine Phosphate, clean-in-place test with 0.2 M NaOH — Figure3CIP condition : 0.2mol/L NaOH 3CV 0.05M phosphate buffer, pH7 5CV (repeat)

Purification data of DNA-binding protein RusA D70N with Cellufine Phosphate — Figure4Use of Cellufine Phosphate in Rus A D70N purification

Chromatography

Column: 1.6x10cm (20ml) packed with Cellufine Phosphate
Flow rate: 3ml/min( 90cm/h )
Sample: 7.5mg of RusA D70N obtained after Heparin-Sepharose chromatography
Gradient: 200ml from 0.1 to 1.3M NaCl in 50mM tris-HCl pH 8.0

SDS-PAGE

Gel: Novex 4-12%BT gel used with MES-SDS running buffer (Invitrogen)
Key for gel: 1 Cell free extract
2 Unbound material from Heparin-Sepharose column
3 RusA sample obtained from Heparin-Sepharose column
4-6 Fractions across the peak eluted from Cellufine Phosphate
7 RusA reference sample
8 Mark 12 MW standard (Invitrogen)

Ref.
Nucleic Acids Research, 2006, Vol. 00, No. 00 1–8
Rachel Macmaster, Svetlana Sedelnikova, Patrick J. Baker, Edward L. Bolt1,Robert G. Lloyd1 and John B. Rafferty
RusA Holliday junction resolvase: DNA complexstructure—insights into selectivity and specificity

This data was carried by courtesy of Dr. Svetlana Sedelnikovaof the Sheffield university.

Protein separation pattern of Cellufine Phosphate — Figure5Separation of mixed sample

Column Size: ID 1.1 cm – Height 10 cm
Flow rate: 2 ml/min (126cm/h)
Buffer: 0.01M acetate buffer, pH4.8
Elution: 0 to 1 mol/L NaCl gradient

Cellufine^TMPhosphate

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