Protein purification by affinity chromatography relies primarily on the interaction of a fused-tag to the protein of interest. Here, we describe a tag-free affinity method that employs functional selection interactions to a broad range of proteins.
Trang 1Muhammad Tehseen1, Vlad-Stefan Raducanu1, Fahad Rashid, Afnan Shirbini,
Masateru Takahashi, Samir M Hamdan∗
King Abdullah University of Science and Technology, Division of Biological and Environmental Sciences and Engineering, Thuwal, 23955, Saudi Arabia
a r t i c l e i n f o
Article history:
Received 2 February 2019
Received in revised form 1 June 2019
Accepted 3 June 2019
Available online 8 June 2019
Keywords:
PCNA
Affinity chromatography
Okazaki fragment
Elution analysis
DNA polymerase
DNA replication
a b s t r a c t Proteinpurificationbyaffinitychromatographyreliesprimarilyontheinteractionofafused-tagtothe proteinofinterest.Here,wedescribeatag-freeaffinitymethodthatemploysfunctionalselection interac-tionstoabroadrangeofproteins.Toachievethis,wecoupledhumanDNA-clampproliferatingcellnuclear antigen(PCNA)thatinteractswithoveronehundredproteinstoanagaroseresin.Wedemonstratethe versatilityofourPCNA-AgarosecolumnatvariouschromatographicstepsbypurifyingPCNA-binding proteinsthatareinvolvedinDNAReplication(DNApolymerase␦,flapendonuclease1andDNAligase1), translesionDNAsynthesis(DNApolymeraseseta,kappaandiota)andgenomestability(p15).Wealso showthecompetenceofthePCNA-Agarosecolumntopurifynon-PCNAbindingproteinsbyfusingthe PCNA-bindingmotifofhumanp21asanaffinitytag.Finally,weestablishthatourPCNA-Agarosecolumn
isasuitableanalyticalmethodforcharacterizingthebindingstrengthofPCNA-bindingproteins.The conservationandhomologyofPCNA-likeclampswillallowfortheimmediateextensionofourmethod
tootherspecies
©2019TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND
license(http://creativecommons.org/licenses/by-nc-nd/4.0/)
∗ Corresponding author.
E-mail address: Samir.hamdan@kaust.edu.sa (S.M Hamdan).
1 These authors contributed equally to the work.
Lig-https://doi.org/10.1016/j.chroma.2019.06.008
0021-9673/© 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.
Trang 2Fig 1.PCNA coupling to various resins(A)Schematic illustration of irreversible coupling of PCNA (N107C-PCNA) with SulfoLink Coupling resin Model of human PCNA was generated using UCSF Chimera from PDB code 1AXC [ 43 ] PCNA subunits are shown in ribbon form in green, yellow and blue The asparagine 107 residue mutation to cysteine is shown in red for one PCNA monomer.(B)Bar chart illustrating the percentage of flow-through, wash and bound fractions of PCNA immobilized through various non-covalent (via Flag and Strep tag) and covalent (via NHS and SulfoLink) chemistries All percentages were calculated from an initial protein amount of 24 mg PCNA and the coupling was performed on 1 ml of each resin as described in the Methods section (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
Trang 3column
c
c 0
UV (c) dc
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⎪
UV (c; h, , , ) =h
2 exp
1 2
2
−c−
erfc
1
√ 2
−c−
UV (c; h, , , ) = hexp
−1 2
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Trang 4
by:
2
2(Table1).Eluentswereconcentratedto1.5ml andthen
(Table1)(Fig.2B,lane3).ThefractionsthatcontainedallPol␦
Fig 2.Purification of human recombinant Pol␦ from Sf9 insect cells.(A)A simpli-fied procedure for purification of Pol␦.(B)SDS-PAGE gel showing different steps
of purification: Lane 1, lysate; lane 2, flow-through from HisTrap; lane 3, protein eluted from HisTrap; lane 4, flow-through from PCNA-Agarose column; lane 5, Pol␦ elution from PCNA-Agarose column; lane 6, Pol␦ after gel filtration All protein frac-tions were separated on a 10% SDS-PAGE gel and stained with Coomassie blue Size marker (M) (kDa) is on the left side of the gel (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table2).ThepurifiedPol␦complexwaselutedasasinglepeakon
column
Trang 5Table 1
Buffers and their composition used in different purification steps (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
11 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 30 mM Imidazole, 10 mM -Mercaptoethanol, and 5% Glycerol.
12 50 mM Tris-HCl (pH 8.0), 160 mM NaCl, 30 mM Imidazole, 10 mM -Mercaptoethanol, and 5% Glycerol.
13 50 mM Tris-HCl (pH 8.0), 160 mM NaCl, 500 mM Imidazole, 10 mM -Mercaptoethanol and 5% Glycerol.
15 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 20 mM imidazole, 10 mM -Mercaptoethanol, and 5% Glycerol.
Table 2
Summary of the fitting parameters of the elution peaks of the purified proteins from the PCNA-Agarose column using the EMG model.
mL
andp15werepreparedasdescribedaboveforPCNA.The
super-natantswerethendirectlyloadedontothePCNA-Agarosecolumn
pre-equilibratedwithbuffer8(Table1)ataflowrateof1ml/min
at ˜340mMNaCl(Figs.6AandB,S1DandTable2).Inthesecond
(Table1)inthepresenceofSUMOproteasetoremovetheSUMO
B,S1FandTable2)
12(Table1)toreducethesaltconcentration.Theboundprotein
Trang 6Fig 3. Purification of human recombinant FEN1, Lig1 and p15 from E coli.(A)A simplified procedure for the purification of FEN1.(B)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from PCNA-Agarose column; lane 3, FEN1 elution from PCNA-Agarose column; lane 4, FEN1 after gel filtration.(C)
A simplified procedure for the purification of Lig1.(D)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from PCNA-Agarose column; lane 3, Lig 1 elution from PCNA-Agarose column; lane 4, flow-through from HiTrap Blue, lane 5, lig1 after HiTrap Blue elution.(E)A simplified procedure for the purification of p15.(F)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from PCNA-Agarose column; lane 3, p15 eluted from PCNA-Agarose column; lane 4, flow-through from HisTrap; lane 5, p15 eluted from HisTrap; lane 6, p15 after SUMO protease digestion; lane 7, untagged p15 in flow-through from HisTrap; lane 8, proteins eluted from HisTrap containing SUMO protease All proteins were separated on a 10% SDS-PAGE gel and stained with Coomassie blue Size markers (M) (kDa) are on the left side of each gel (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
B,S1AandTable2).Thepeakfractionswerecollectedanddialyzed
Trang 7Fig 4. Purification of human recombinant translesion DNA polymerases (Pol, Pol, Pol) from E coli.(A)A simplified procedure for the purification of Pol.(B)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from HisTrap; lane 3, Pol eluted from HisTrap; lane 4, flow-through from PCNA-Agarose column; lane 5, Pol eluted from PCNA-Agarose column; lane 6, Pol after SUMO protease digestion; lane 7, untagged Pol in flow-through from HisTrap; lane 8, proteins eluted from HisTrap containing SUMO protease; lane 9, Pol after gel filtration.(C)A simplified procedure for the purification of Pol.(D)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from HisTrap; lane 3, Pol eluted from HisTrap; lane 4, flow-through from PCNA-Agarose column; lane 5, Pol eluted from PCNA-Agarose column; lane 6, Pol after SUMO protease digestion; lane 7, untagged Pol in flow-through from HisTrap; lane 8, proteins eluted from HisTrap containing SUMO protease.(E)A simplified procedure for the purification of Pol.(F)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from HisTrap; lane 3, Pol eluted from HisTrap; lane 4, Pol after SUMO protease digestion; lane 5, untagged Pol in flow-through from HisTrap; lane 6, proteins eluted from HisTrap containing SUMO protease; lane 7, flow-through from PCNA-Agarose column; lane 8, Pol eluted from PCNA-Agarose column All proteins were separated on a 10% SDS-PAGE gel and stained with Coomassie blue Size markers (M) (kDa) are on the left side of each gel (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
(Table2)
clamp
Trang 8Fig 5. Purification of PIP p21 -Tus from E coli.(A)Schematic representation of recombinant PIP p21 -Tus expression construct.(B)A simplified procedure for the purification
of PIP p21 -Tus.(C)SDS-PAGE gel showing the different steps of purification: Lane 1, lysate; lane 2, flow-through from HisTrap; lane 3, Tus eluted from HisTrap; lane 4, flow-through from PCNA-Agarose column; lane 5, Tus elution from PCNA-Agarose column; lane 6, Tus after SUMO protease digestion; lane 7, untagged Tus in flow-through from HisTrap; lane 8, proteins eluted from HisTrap containing SUMO protease All protein fractions were separated on a 10% SDS-PAGE gel and stained with Coomassie blue Size markers (M) (kDa) are on the left side of each gel (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig 6. Analysis of the elution chromatograms of the purified proteins from the PCNA-Agarose column.(A)Plot of the cumulative elution percentage versus the NaCl concentration for each of the studied proteins The cumulative elution percentage is obtained as described in the Methods section The color of the curves corresponds to those indicated in the inset table The horizontal dashed line (red) indicates 50% cumulative elution The intersection of each cumulative elution percentage curve gives the median NaCl concentration of elution for each protein The median values are recorded in the inset table.(B)Corresponding concentrations of the maxima of the elution peaks for each studied protein as described in the Methods section The values are obtained by fitting the elution peaks using the described EMG model (Fig S1) The vertical bars indicate the 95% confidence interval for the positions of the maxima of the elution peaks The bars corresponding to the proteins known to contain a single PIP box are colored in red, while those corresponding to the proteins containing multiple PIP boxes are colored in blue.
byLig1(Fig.6A,BandTable2).Thisorderofaffinityisnot
frag-mentFEN1andLig1(Fig.6A,BandTable2).PolhasonlyonePIP
column
Trang 9manuscript
Funding
06.008
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