Reducing DNA content during protein purification with an...
1
• HL-SAN efficiently digests DNA in high salt His-Trap eluates containing imidazole • The lowest DNA contamination in the final product is achieved by HL-SAN treatment of both the lysate and the eluate • HL-SAN is efficiently separated from T4 ligase product by cation exchange chromatography • T4 ligase activity is unaffected by HL-SAN treatment Conclusions Contact info: [email protected] PepTalk 2015, San Diego USA, January 19—23 T4 Ligase is unaffected by HL-SAN treatment 4 - Final Product #1a #1b #2a #2b 18-bp » 9-bp » Figure 4: DNA ligase activity was assayed by incubation of 80 nM of a singly-nicked double-stranded substrate with 0.6 μM T4 product for 15 min at 25°C (0.1 mM ATP, 10mM MgCl 2 , 1mM DTT 100 mM NaCl, 50 mM Tris pH 8.0). The ligated band (18-bp) and un-ligated bands (9-bp) were resolved by denaturing gel electro- phoresis (20% acrylamide, 7M urea, 1x TBE gel) and visualized by excitation of a fluorescent label. No difference in the specific activity of the concentration-normalized T4 ligase was seen with HL-SAN treatment, and no degradation of assay substrates due to residual nuclease activity was detected. HL-SAN is removed by cation exchange chromatography #1a #1b #2a #2b #1a #1b #2a #2b #1a #1b #2a #2b NC M HL-SAN in Lysate HL-SAN in His Eluate Sample Marker Neg Control 2 His-Trap eluate 3 Dialyzed 4 SP Flow-through — + + + — + — — — + + + — + — — — + + + — + — — Nicked » Supercoiled » Partially degraded » HL-SAN reduces DNA in His-trap eluate 0 5 10 15 20 25 30 35 40 45 Fluorescence (dR) Cycles Untreated eluate (#1b) + HL-SAN in eluate (#1a) 3 Untreated lysate Untreated eluate (#2b) + HL-SAN in eluate (#2a) 3 HL-SAN in lysate NTC 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 M #1 #2 #1 #2 Marker 1 Untreated Lysate 1 Lysate + HL-SAN 2 His FT, No SAN 2 His FT, lys + SAN 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 0 5 10 15 20 25 30 35 40 45 Fluorescence (dR) Cycles Untreated (#1) + HL-SAN (#2) 2 HisTrap FT NTC Fig.1A Fig.1B HL-SAN reduces DNA in the crude lysate Results Figure 1A: HL-SAN greatly reduced DNA content of the crude lysate, shown by agarose gel electrophoresis of undiluted lysate and His-trap flow-through, and in both cases the majority of the DNA remained in the flow-through. Figure 1B: E. coli 23S DNA in flow-through was quantified by qPCR, and it was determined that HL-SAN treatment of the lysate reduced DNA content about 10,000 fold. Figure 3: HL-SAN was highly active in His-trap eluate, where 1 μl of sample completely digested 25 μl double stranded plas- mid DNA (45 mM Tris pH 8, 20 mM MgCl 2 ) after 2 hours at 37ºC. The majority of this activity was removed by passing eluate over an SP sepharose column, after which only minor DNA nicking was observed, corresponding to the background EndA activity from the BL21 host-cell strain. Figure 2: Although most of the DNA was removed by the His-Trap, some is still present in the eluate. HL-SAN treatment of the lysate reduced DNA in the flow-through 10,000 fold, however the reduction in the eluate was only about 200-fold. A similar reduction was achieved by direct HL-SAN treatment of the eluate alone, while combining HL-SAN treatment of both lysate and eluate resulted in about 4,000-fold reduction in DNA as compared to untreated sample. Experimental setup T4 Ligase expression (E. coli BL21) Lysis (French press) His-Trap Chromatography Dialysis SP Chromatography #1 #2 #1b #1a #2b #2a HL-SAN treatment Crude lysate 1 U HL-SAN per mg cell pellet +10 mM MgCl 2 1 hr at 4°C HL-SAN treatment His-Trap Eluate 2000 U HL-SAN per ml eluate +10 mM MgCl 2 1 hr at 15°C HL-SAN Removal SP Sepharose binding Tris pH 8.0 1 mM DTT 1 Crude Lysate 2 Eluate 3 Dialyzed 4 Flow-through: Pure T4 Ligase 750 mM NaCl 750 mM NaCl ~300 mM imidazole 200 mM NaCl HL-SAN HL-SAN HL-SAN Salt Content When purifying DNA-modifying enzymes, an essentially DNA-free product is desirable as endogenous DNA from production may contaminate down-stream applications. While nuclease treatment of the crude cell-lysate is a common step during protein purification, its primary function is viscosity reduction for easier sample handling rather than complete DNA remov- al. More efficient enzymatic DNA removal could be achieved if applied to a partially purified protein sample, such as the His-trap eluate, where the majority of impurities have been removed and the volume is reduced; however the salt concentrations used in elution protocols are incompatible with the salinity optima of most commercially available nucleases. Furthermore, treatment of semi-pure protein may cause residual nuclease contamination in the purified protein which will interfere with nucleic-acid based applications. ArcticZymes has developed a Heat-Labile Salt Active Nuclease (HL-SAN) that is compatible with treat- ment of eluates: it is active at up to 1M NaCl, tolerates imidazole and can be separated from most pro- teins by cation exchange or inactivated by reducing agents. Here we present efficient DNA removal from recombinantly-produced T4 DNA ligase by direct treatment of the His-trap eluate with HL-SAN, and demonstrate that the final, active T4 ligase is free of contaminating HL-SAN activity. Introduction Reducing DNA content during protein purification with an easily removable nuclease Bernd Striberny 1,2 , Jørn Remi Henriksen 1 , Morten Elde 1 , Olav Lanes 1 , Adele Williamson 2 1. ArcticZymes AS, Tromsø, Norway 2. Department of Chemistry, NorStruct, UiT - The Arctic University of Norway, Tromsø, Norway
Transcript of Reducing DNA content during protein purification with an...
• HL-SAN efficiently digests DNA in high salt His-Trap eluates containing imidazole
• The lowest DNA contamination in the final product is achieved by HL-SAN treatment of both the lysate and the eluate
• HL-SAN is efficiently separated from T4 ligase product by cation exchange chromatography
• T4 ligase activity is unaffected by HL-SAN treatment
Conclusions
Contact info: [email protected] PepTalk 2015, San Diego USA, January 19—23
T4 Ligase is unaffected by HL-SAN treatment
4 - Final Product
#1a #1b #2a #2b
18-bp »
9-bp »
Figure 4: DNA ligase activity was assayed by incubation of 80
nM of a singly-nicked double-stranded substrate with 0.6 µM T4
product for 15 min at 25°C (0.1 mM ATP, 10mM MgCl2, 1mM DTT
100 mM NaCl, 50 mM Tris pH 8.0). The ligated band (18-bp) and
un-ligated bands (9-bp) were resolved by denaturing gel electro-
phoresis (20% acrylamide, 7M urea, 1x TBE gel) and visualized
by excitation of a fluorescent label. No difference in the specific
activity of the concentration-normalized T4 ligase was seen with
HL-SAN treatment, and no degradation of assay substrates due
to residual nuclease activity was detected.
HL-SAN is removed by cation exchange chromatography#1a #1b #2a #2b #1a #1b #2a #2b #1a #1b #2a #2b NC M
HL-SAN in Lysate
HL-SAN in His Eluate
Sample
Marker
Neg
Co
ntrol2 His-Trap eluate 3 Dialyzed 4 SP Flow-through
— + +
+
—
+ — —
— + +
+
—
+ — —
— + +
+
—
+ — —
Nicked »
Supercoiled »
Partially degraded »
HL-SAN reduces DNA in His-trap eluate
0 5 10 15 20 25 30 35 40 45
Fluo
resc
ence
(dR
)
Cycles
Untreated eluate (#1b)
+ HL-SAN in eluate (#1a)
3 Untreated lysate
Untreated eluate (#2b)
+ HL-SAN in eluate (#2a)
3 HL-SAN in lysate
NTC0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000M #1 #2 #1 #2
Marker
1 U
ntreated Lysate
1 Lysate +
HL-SA
N
2 H
is FT, No
SAN
2 H
is FT, lys + SA
N
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
0 5 10 15 20 25 30 35 40 45
Fluo
resc
ence
(dR
)
Cycles
Untreated (#1)
+ HL-SAN (#2)
2 HisTrap FT
NTC
Fig.1A Fig.1B
HL-SAN reduces DNA in the crude lysate
Results
Figure 1A: HL-SAN greatly reduced DNA content of the crude lysate, shown by agarose gel electrophoresis of undiluted
lysate and His-trap flow-through, and in both cases the majority of the DNA remained in the flow-through.
Figure 1B: E. coli 23S DNA in flow-through was quantified by qPCR, and it was determined that HL-SAN treatment of the
lysate reduced DNA content about 10,000 fold.
Figure 3: HL-SAN was highly active in His-trap eluate, where 1 µl of sample completely digested 25 µl double stranded plas-
mid DNA (45 mM Tris pH 8, 20 mM MgCl2) after 2 hours at 37ºC. The majority of this activity was removed by passing eluate
over an SP sepharose column, after which only minor DNA nicking was observed, corresponding to the background EndA
activity from the BL21 host-cell strain.
Figure 2: Although most of the DNA was removed by the His-Trap, some is still present in the eluate. HL-SAN treatment
of the lysate reduced DNA in the flow-through 10,000 fold, however the reduction in the eluate was only about 200-fold.
A similar reduction was achieved by direct HL-SAN treatment of the eluate alone, while combining HL-SAN treatment of
both lysate and eluate resulted in about 4,000-fold reduction in DNA as compared to untreated sample.
Experimental setupT4 Ligase expression
(E. coli BL21)
Lysis (French press)
His-Trap Chromatography
Dialysis
SP Chromatography
#1 #2
#1b#1a #2b#2a
HL-SAN treatmentCrude lysate1 U HL-SAN per mg cell pellet+10 mM MgCl2
1 hr at 4°C
HL-SAN treatmentHis-Trap Eluate2000 U HL-SAN per ml eluate+10 mM MgCl2
1 hr at 15°C
HL-SAN RemovalSP Sepharose bindingTris pH 8.01 mM DTT
1 Crude Lysate
2 Eluate
3 Dialyzed
4 Flow-through: Pure T4 Ligase
750 mM NaCl
750 mM NaCl~300 mM imidazole
200 mM NaCl
HL-SANHL-SAN
HL-SANSalt Content
When purifying DNA-modifying enzymes, an essentially DNA-free product is desirable as endogenous DNA from production may contaminate down-stream applications.
While nuclease treatment of the crude cell-lysate is a common step during protein purification, its primary function is viscosity reduction for easier sample handling rather than complete DNA remov-al. More efficient enzymatic DNA removal could be achieved if applied to a partially purified protein sample, such as the His-trap eluate, where the majority of impurities have been removed and the volume is reduced; however the salt concentrations used in elution protocols are incompatible with the salinity optima of most commercially available nucleases. Furthermore, treatment of semi-pure protein may cause residual nuclease contamination in the purified protein which will interfere with nucleic-acid based applications.
ArcticZymes has developed a Heat-Labile Salt Active Nuclease (HL-SAN) that is compatible with treat-ment of eluates: it is active at up to 1M NaCl, tolerates imidazole and can be separated from most pro-teins by cation exchange or inactivated by reducing agents. Here we present efficient DNA removal from recombinantly-produced T4 DNA ligase by direct treatment of the His-trap eluate with HL-SAN, and demonstrate that the final, active T4 ligase is free of contaminating HL-SAN activity.
Introduction
Reducing DNA content during protein purification with an easily removable nucleaseBernd Striberny1,2, Jørn Remi Henriksen1, Morten Elde1, Olav Lanes1, Adele Williamson2
1. ArcticZymes AS, Tromsø, Norway2. Department of Chemistry, NorStruct, UiT - The Arctic University of Norway, Tromsø, Norway
