Chromatography
Analysis
Application Note: CHROM302
Purification of Staphylococcal Surface Protein Domains
on OmniSep™ A-Ni Dash and a Leading Nickel AgaroseBased Media
Introduction
In the early stages of infection, bacteria such as Staphylococcus aureus attach to host tissue via proteins. Interactions between
host and bacterial proteins are also likely to play an important role in the maintenance and dissemination of infection.
Understanding the mechanisms of host-pathogen interactions is a key area for current research.
Most recombinant proteins may be cloned and expressed in E.coli. The use of E.coli for protein expression is well documented for
its advantages of low cost, fermentation and ease of transformation, and high protein yields. However, solubility may be an issue,
since some proteins are insoluble and aggregate in inclusion bodies. The following factors can affect expression levels and/or
solubility: growing temperature, concentration of inducer (IPTG), host strain, protein size and structure, and toxicity.
Method
The same conditions were employed for both the OmniSep A-Dash Ni 5ml and a leading nickel agarose-based 5ml column. Two
(2) 5 ml columns were attached together to operate in tandem.
Binding buffer A: 20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5
Elution buffer B: 20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5
All purifications were performed using either an Akta PrimePlus or an Akta Purifier10.
Proteins 9, 11, 12 and 13 were expressed in strains of E.coli which are the standard BL21 (DE3) strains initially developed by the
Brookhaven National Laboratories and purified from the soluble fraction of the cell lysate.
The columns were equilibrated in binding buffer A. The lysate was passed through the column at a flow rate 2-5 ml/min and the
flow-through is collected (FT). The columns was then washed (W) with binding buffer until the absorbance (at 280 nm) of the
solution eluting from the column was <50 mAu and stable.
The proteins of interest were then eluted in individual runs using a gradient of 0 to 50% elution buffer B over 20 column volumes.
Appropriate fractions were collected for each protein purified and analysed using SDS-PAGE, along with the FT and W.
SDS-PAGE gels (10% polyacrylamide) and all buffers were made internally. Electrophoresis was performed at 200V for 45 minutes.
The gels were then stained using Coomassie dye and destained using ethanol/acetic acid.
Gel nomenclature: FT = flow-through, W = wash, S = total soluble protein before chromatography, I = insoluble protein, L = ladder
(Bio-Rad Precision Plus Protein™ All Blue Standards was employed), the numbers for specific fractions collected relate to the
associated chromatogram.
Results
The results are presented as comparative chromatograms generated from the ÄKTA® instruments along with SDS-PAGE gels. It
can be seen that equivalent separations for all proteins are obtained for the OmniSep A-Ni Dash columns and the leading nickel
agarose-based columns. In some cases from a chromatographic perspective the separations on the OmniSep columns is superior,
eluting as a sharper peak in a smaller volume.
OmniSep Protein 9
S
I FT/W L 8
10 12 14 16 18 20 22
Figure 1. SDS-PAGE for fractions of Protein 9 from the OmniSep A-Ni Dash column
Leading Nickel Protein 9
FT/W L 8 10 12 14 16 18 20 22 24 26 28
Figure 2. SDS-PAGE for fractions of Protein 9 from a leading nickel agarose-based column
2013May22no002:101_UV
mAu
2013May22no002:101_Cond
2013May22no002:101_Conc
OmniSep A-Ni Dash Column
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0
50
2013May22no001:10_UV
mAu
100
150
2013May22no001:10_Cond
200
ml
2013May22no001:10_Conc
Leading Nickel Agarose-Based Column
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0
50
100
150
200
ml
Figure 3. Comparison Chromatograms for the purification of Protein 9 on a 5ml OmniSep A-Ni Dash column (top) and a
5ml leading nickel agarose-based column (bottom).
Leading Nickel Potein 11
S FT 8 12 16 20 24
L
OmniSep Protein 11
S FT 8 12 16 20
Figure 4. SDS-PAGE for fractions of Protein 11 from both the OmniSep A-Ni Dash and a
leading nickel agarose-based column
2013May28no002:101_UV
mAu
120
2013May28no002:101_Cond
2013May28no002:101_Conc
OmniSep A-Ni Dash Column
100
80
60
40
20
0
-20
-40
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0
50
2013May28no001:10_UV
mAu
100
150
2013May28no001:10_Cond
200
ml
2013May28no001:10_Conc
Leading Nickel Agarose-Based Column
200
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0
50
100
150
200
ml
Figure 5. Comparison Chromatograms for the purification of Protein 11 on a 5ml OmniSep A-Ni Dash column (top) and a
5ml leading nickel agarose-based column (bottom).
Leading Nickel Protein 12
S
FT 8 12 16 20 24
L
OmniSep Protein 12
S FT 8 12 16 20
Figure 6. SDS-PAGE for fractions of Protein 12 from both the OmniSep A-Ni Nash and a
leading nickel agarose-based column
2013May29no001:10_UV
2013May29no001:10_Cond
2013May29no001:10_Conc
OmniSep A-Ni Dash Column
mAu
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0.0
10.0
2013May29no003:101_UV
mAu
20.0
30.0
2013May29no003:101_Cond
40.0
min
2013May29no003:101_Conc
Leading Nickel Agarose-Based Column
200
150
100
50
0
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
50
100
150
200
ml
Figure 7. Comparison Chromatograms for the purification of Protein 12 on a 5ml OmniSep A-Ni Dash column (top) and a
5ml leading nickel agarose-based column (bottom).
Leading Nickel Protein 13
S
FT
8
12
16
OmniSep Protein 13
20
24
L
S
FT
8
12
16
Figure 8. SDS-PAGE for fractions of Protein 13 from both the OmniSep A-Ni Nash and a
leading nickel agarose-based column
mAu
2013May29no002:101_UV
2013May29no002:101_Cond
2013May29no002:101_Conc
OmniSep A-Ni Dash Column
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0
50
2013May29no004:10_UV
mAu
200
100
150
2013May29no004:10_Cond
200
ml
2013May29no004:10_Conc
Leading Nickel Agarose-Based Column
150
100
50
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51
0
50
100
150
200
ml
Figure 9. Comparison Chromatograms for the purification of Protein 13 on a 5ml OmniSep A-Ni Dash column (top) and a
5ml leading nickel agarose-based column (bottom).
Trademarks
OmniSep™ is a trademark of Diba Industries Incorporated.
ÄKTA™ is a trademark of GE Healthcare Life Sciences.
Available worldwide through
UNITED KINGDOM
Kinesis Ltd
Tel: +44 (0)1480 212122
Fax: +44 (0)1480 212111
E-mail: sales@kinesis.co.uk
Web: kinesis.co.uk
GERMANY, SWITZERLAND
& AUSTRIA
Kinesis GmbH (formerly Abimed)
Tel: +49 (0)2173 89 05-0
Fax: +49 (0)2173 89 05-77
Email: sales@kinesisgmbh.de
Web: kinesisgmbh.de
Manufactured by
USA & CANADA
Kinesis Inc
Tel: (518) 289-5817
Toll free in USA: (866) 934-6353
Fax: (518) 289-5818
Email: sales@kinesis-usa.com
Web: kinesis-usa.com
AUSTRALIA & NEW ZEALAND
Kinesis Australia Pty Ltd
Tel: +61 (0)7 3829 3996
Fax: +61 (0)7 3829 3997
Email: sales@kinesis-australia.com.au
Web: kinesis-australia.com.au
EUROPE
Diba Industries Ltd.
2 College Park
Coldhams Lane
Cambridge, CB1 3HD, UK
Tel: +44 (0) 1223 472801
Fax: +44 (0) 1223 416787
sales@dibaind.com
www.omnisep.com
www.dibaind.com