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Efficient, ultra-high-affinity chromatography in a
one-step purification of complex proteins
Marina N. Vassylyevaa^ , Sergiy Klyuyev a^ , Alexey D. Vassylyev a^ , Hunter Wesson a, Zhuo Zhang a, Matthew B. Renfrow a^ , Hengbin Wanga^ , N. Patrick Higgins a, Louise T. Chow a,1^ , and Dmitry G. Vassylyev a,
aDepartment of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294
Contributed by Louise T. Chow, April 25, 2017 (sent for review March 24, 2017; reviewed by Robert Landick and Tahir H. Tahirov)
Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactom- ics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10 −^14 – 10 −^17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We dem- onstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays.
Colicin E7/Im7 affinity chromatography | one-step protein purification |
RNA polymerases | membrane proteins | condensin
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btaining protein samples with high yield, high purity, and high activity (HHH purification) is the foundation for most
Appendix, Fig. S2B). Consistently, we obtained similar results with other low-salt systems, including GST (GST-Tag; SI Appendix, Fig. S2C) and streptavidin-binding peptide (Strep-Tag; noted in SI Appendix, Fig. S2) columns. Thus, a one-step HHH purification using these “low-salt” columns is limited to only a subset of bi- ological targets that do not have nonspecific interactions with untagged molecules (proteins, DNA/RNA) under the low-salt conditions (see examples in SI Appendix, Fig. S3 B and C). Only the chitin-binding (ChBD) and His-Trap affinity systems tolerate high salt-containing buffers (∼1 M NaCl) at the crucial step of crude lysate loading onto the columns (Fig. 1A). How- ever, the chitin columns have a low capacity, and in our experi- ence, some contaminants always remained in the purified samples (SI Appendix, Fig. S4). Accordingly, this approach is rarely used for crystallographic studies (4) (SI Appendix, Figs. S5 and S6). To date, His-Trap (Ni^2 +-based) is the most com- monly used approach. It demonstrates a high-yield capacity and also allows for high-salt loading of the lysates without losing binding affinities (Fig. 1A). Additional advantages include the low costs of the Ni 2 +-charged beads (SI Appendix, Fig. S1A) that can be put through multiple regeneration cycles, the small size of the His-tag, and efficient target elution with the inexpensive imidazole. However, our results and those of other investigators show that this approach possesses several drawbacks. It has relatively high, nonspecific, but cooperative affinity to DNA and to DNA-binding proteins (5) as well as to some cellular proteins (4), including many membrane (6, 7) and eukaryotic proteins (8).
Significance
Protein purification is a primary step and the basis for numerous biochemical and biomedical studies. It is particularly crucial for high-resolution structural analysis and industrial protein pro- duction, where it has to meet the high-yield, high-purity, and high-activity (HHH) requirement. However, the HHH purification of many proteins or protein complexes remains a difficult, target-dependent, and multistep process. The ultra-high-affinity (CL7/Im7) purification system described in this work allows for one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, mem- brane, toxic, and DNA/RNA-binding proteins and complexes. It might emerge as an efficient, universal tool for high- throughput isolation of many significant biological systems to advance modern biological studies as well as manufacturing of therapeutic proteins.
Author contributions: H. Wang and D.G.V. designed research; M.N.V., S.K., A.D.V., H. Wesson, Z.Z., M.B.R., H. Wang, N.P.H., and D.G.V. performed research; D.G.V. contrib- uted new reagents/analytic tools; M.N.V., M.B.R., H. Wang, N.P.H., L.T.C., and D.G.V. analyzed data; and L.T.C. and D.G.V. wrote the paper. Reviewers: R.L., University of Wisconsin–Madison; and T.H.T., Nebraska Medical Center. Conflict of interest statement: The CL7/Im7 purification technology is protected by a PCT patent (PCT/US2016/065843) filed by the UAB Research Foundation. (^1) To whom correspondence may be addressed. Email: ltchow@uab.edu or dmitry@ uab.edu.
modern biological studies, such as proteomics, interactomics, and in vitro drug screening. In most cases, it also raises such studies to new heights. Moreover, HHH-grade samples are in- dispensable for industrial production of therapeutic proteins and for determining high-resolution 3D protein structures that are crucial for deep understanding of protein function. In recent years, investigations of large multisubunit complexes (transcrip- tion and translation machineries, for example) and challenging eukaryotic and membrane proteins have emerged as a major focus of proteomics and structural analyses. An efficient, ideally one-step protocol for HHH purification is highly desirable to facilitate all these crucial studies. Commercial purification systems have complementary advan- tages that allow the purification of target proteins (1–3). However, each has certain critical disadvantages that affect their efficiencies (1–3). Some columns have low capacity and often command a high price (SI Appendix, Fig. S1A) such that they barely meet the re- quirement of high yield necessary for structural studies or for commercial protein production. Salt sensitivity is another notable limitation of many (protein/ligand) affinity systems; it often results in significant flow through (90–95%) of the tagged proteins even when medium salt (0.2–0.5 M) is used during lysate loading on the columns (Fig. 1 A). On the other hand, if the sample is loaded in a low-salt buffer, impurities often could not be removed afterward even when very high-salt buffer is applied. This salt dilemma is well illustrated by our purification trials with the two DNA- binding proteins fused to the maltose-binding protein (MBP- Tag; SI Appendix, Fig. S2 A and B). Importantly, these results demonstrate that even loading buffer with 0.5 M salt is not suffi- cient to obtain high purity for some DNA-binding proteins (SI
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These contaminants occur largely in a salt-independent manner, sometimes resulting in substantial impurities in the recovered target protein. For instance, according to the combined statistics of the Structural Genomics Projects, all of which use the His-Trap technique as the first purification step, additional purification steps were always required to obtain high-purity samples (4), sig- nificantly compromising their yields and increasing purification time. In addition, according to the commercial protocols, the His- Trap Ni^2 +^ base cannot resist metal chelating and reducing agents at concentrations that are required to maintain physiological properties of the certain categories of target proteins. To gain a more detailed insight into the present state of pu- rification of complex proteins, we have analyzed the published protocols for a number of biologically significant membrane and DNA-binding proteins, for which high-resolution crystal struc- tures have been determined (SI Appendix, Figs. S5 and S6). The purification of these proteins is usually difficult, especially for crystallographic purposes, and may serve as a benchmark that a newly devised HHH purification strives to exceed. The analysis revealed several general observations. First, in agreement with the major advantages of the His-Trap approach, it was used as a first-step purification in the overwhelming majority (∼80%) of the studies (Fig. 1A). Other affinity approaches were used as a first step only in a few (∼8%) cases overall and only in 3% of cases of DNA-binding proteins, a reflection of their relatively poor capacities or “low-salt loading” limitations (Fig. 1A). Sec- ond, none of purifications could be completed in one step, consistent with the above noted disadvantages of the His-Trap and other affinity techniques. In fact, an average purification protocol includes ∼3 chromatographic steps and takes over 3 d at best. Third, most protocols include one or more nonspecific columns [gel filtration (GF), ion exchange, nucleic acid (NA) mimicking resins] that are usually substantially less predictable than those based on specific affinity. As a result, additional ef- forts are required to customize the conditions for each particular protein. Finally, even the simplest, two-step (His-Trap → GF) protocol (∼10% of analyzed studies) takes at least 2 d and typ- ically have protein loss (∼50%) during the GF step (SI Appendix, Figs. S5 and S6). In conclusion, the HHH-grade purification of challenging protein or protein complexes remains a target-dependent and multistep process. The biochemical and biomedical field would greatly benefit from a new approach, which combines the major advantages of the His-tag technique (high capacity, high-salt tol- erance, and low cost) with a stringent protein/ligand-based affinity system. Here we report the development of an original, ultra-high- affinity purification system based on the small protein/protein complex, Colicin E7 DNase (∼16 kDa) and Immunity Protein 7 (∼10 kDa) (CE7/Im7) (9). With a Kd ∼ 10 −^14 – 10 −^17 M, the af- finity of this complex is approaching that of a covalent bond and is 4 – 7 orders of magnitude higher than those of any other available analogs (1, 10–16) (Fig. 1A). Because the DNase activity of CE7 is lethal to the cells, we designed a catalytically inactive variant, termed CL7, based on molecular modeling. We also engineered an Im7 immobilization unit to allow efficient coupling to agarose beads without compromising its association with the CL7 tag. We demonstrate that, unlike other approaches, this CL7/Im7 system allows for a one-step HHH purification (∼ 97 – 100% purity) of the most challenging biological targets, including large multisubunit complexes, DNA/RNA-binding proteins, membrane proteins, as well as the proteins poorly expressed in the native host cells. Further, the Im7 affinity column can be easily and repeatedly regenerated without losing its affinity to the CL7 affinity tag.
Results The Design of the CL7/Im7 Chromatographic System. The family of colicin DNases (CE2, CE7, CE8, CE9) belongs to the category of highly toxic proteins because of their DNase domains (∼16 kDa)
Fig. 1. The CL7/Im7 purification system. (A) Comparison of the chromatographic systems. (B) Protocols of the CL7/Im7 purification. (C and D) Purification of the Im7 unit (Im7) (C) and the model CL7M protein (D). ALL, combined statistics for both (MP+NAB) sets; CalBD, calmodulin-binding domain; ChBD, chitin-binding domain; CL7, engineered CE7 tag; first step, a frequency (%), with which a tech- nique was used on the most crucial, first (lysate) purification step in the analyzed sets of the MPs and NABs; FLAG, antibody-binding peptide; Im7F, expression vector for Im7 fusion; MBP, maltose-binding protein; MP/NAB, the analyzed sets of the membrane/NA-binding proteins (SI Appendix, Figs. S5 and S6); Pr-A, protein A tag; Strep, streptavidin-binding peptide. The cleaved IM7 recovered from FT 2 was used for all of the trials in this study. EL, eluate; FT, flow through; Gdn, guanidine hydrochloride; H8, 8 histidine tag; LD, ladder (kDa); LYS, lysate; P(PSC)/P(SMP), PSC/ SMP cleavage sites; PSC, PreScission protease; SM, SMP, SUMO domain and pro- tease; T, target protein; Trx, thioredoxin. The same abbreviations are also used in other figures. *The details of the protocols and Kd values are provided in the legend of SI Appendix, Fig. S1. **At saturation conditions (visible FT), ∼ 400 mg of CL7M is eluted from the 20 mL Im7-column—that is, ∼ 20 mg/1 mL Im7 beads.
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Thus, their expression as a fusion protein would alleviate the loss of the ω subunit in this initial step. In the MV0 construct (Fig. 3, top line), we have introduced only a His-tag at the C terminus of the largest (β’ω) subunit to test the expression and purification performance. The vector demonstrated a modest expression level of the intact RNAP subunit molecules (Fig. 4A, left lane). However, multiple at- tempts under different loading and washing conditions failed to provide a reasonably pure sample even though a heated lysate was used to eliminate most of the E. coli proteins (Fig. 4B). The vector was then modified to relocate the His-tag to the second largest β subunit and to add the CL7-tag (cleavable by PSC) at the C terminus of the β’ω subunit. In addition, we have introduced N-terminal short PSC cleavable “expression” tags (SI
Fig. 2. Comparative performance of the CL7/Im7 system versus conventional protocols. bh, B. halodurans (6, 7); DNC, DNA cellulose; D-PL, dual plasmid expression approach; ec, E. coli (21); GF, gel filtration; Heat, heating at 60 °C; HEP, heparin; His, His-Trap (Ni 2 +); HOST, expression from the host chromosome; IEQ/IES, anion/cation exchange; k, productivity factor [PF = protein amount (PA)/purification time (PT)]; M-PR, multipromoter expression vector; mt, M. tu- berculosis (5); ORG, host organism; P-CYS, single promoter/polycystronic expression vector; PEI/AS, polyethyleneimine/ammonium sulfate precipitations; PF (Im7), PF of the Im7 approach with respect to the other protocols; SPN/SP1, E. coli overexpression using the Native/Engineered (with C/S mutation) YidC signal peptides; taq, T. aquaticus (22); [TEV], TEV protease cleavage; tt, T. thermophilus (28); UCF, ultracentrifugation. PT and PA for the published protocols were estimated based on our own experience with the same or similar protocols.
Fig. 3. Multisubunit expression vectors. E1/E2/E3, the short N-terminal “ex- pression” peptides; P(PSC), PSC cleavage sites; SU, subunit, each has its own ri- bosome binding site and stop codon; T7P, T7 promoter (SI Appendix, Fig. S9B).
studied these RNAPs in the past decade using various tech- niques, including high-resolution crystallographic analysis, for which HHH purification is of central importance (26–29). Our objective, therefore, was not solely to test the purification system but also importantly to establish a simple and straightforward approach for production and HHH purification of RNAPs from different organisms suitable for mutagenesis and successful high- throughput crystallization. To develop an efficient protocol of the large-scale production and HHH isolation of RNAPs, we have designed several multi- subunit, polycystronic expression vectors. Each used a T7 pro- moter inducible by IPTG, and the ORF of each subunit was preceded by a ribosome-binding site. There are two major cri- teria for their designs. The vector should be easily used to clone RNAPs from various species, and it should be applicable to other multisubunit proteins as well. It should also possess enhanced expression levels of the key (or each) individual subunits. We began the vector design using ttRNAP, as it is the most difficult target for overexpression in E. coli. There are no effi- cient expression/purification protocols for recombinant ttRNAP because the T. thermophilus genes have exceedingly high G/C (∼70%) content and contain a high frequency of E. coli rare codons. These features result in overall poor expression levels along with many translational truncations. To minimize these obstacles, we synthesized the genes of the RNAP subunits; the rare codons were eliminated, whereas the GC content was de- creased to a reasonable level of ∼59% for expression in E. coli. To provide a reference point and to compare the new CL7/ Im7 purification system with the most popular His-Trap ap- proach, we created three expression vectors (Fig. 3). In each, we fused the C terminus of the largest β’ subunit to the N terminus of the smallest ω subunit through a flexible (removable by PSC) linker (SI Appendix, Fig. S9A). This approach has two notable advantages. It reduces the number of coexpressed subunits while maintaining a perfect stoichiometry of the β’ and ω subunits during purification. The second is especially important. Our ex- perience with RNAP purifications indicates that the high-salt (∼ 1 M NaCl) loading conditions dissociate these two subunits.
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Appendix, Fig. S9B) designed to increase the expression levels of the two largest β and β’ω-CL7 subunits (Fig. 3, MV1, middle line) or all three (α, β, β’ω-CL7) coexpressed subunits (Fig. 3, MV2, bottom line). As expected, the expression of two (from MV1; Fig. 4A, middle lane) or all (from MV2; Fig. 4A, right lane) RNAP subunits was substantially improved. The production levels are (α > β > β’ω-CL7) from the superior MV2 expression system (Fig. 4C). We were able to obtain a large amount (∼37 mg) of a pure and intact ttRNAP sample in a single Im7 purification step from lysates of 3 g of cells (Fig. 4C). Notably, the best purity of ttRNAP using either the His-Trap or the CL7/Im7 approaches was achieved if the lysates were first treated with DNase before loading onto the columns in high (1–1.2 M) NaCl-containing buffers. Loading the lysates at a lower (0.5–0.8 M) salt concentrations or without prior DNase treatment always produced somewhat contaminated and only partially active products, even when the column was washed with extra-high-salt (2 M) buffers after loading. The activity tests using the synthetic elongation scaffold showed that the recombi- nant ttRNAP was as active as enzyme purified from the host thermophilic cells, which were previously used in successful high- resolution structural studies (26–28) (Fig. 4D). Given the successful one-step HHH purification protocol for ttRNAP, we adjusted the sequence of mtRNAP to the E. coli co- dons and used the identical expression (Fig. 3, MV2) and purifi- cation approaches. We obtained essentially the same results (Fig. 5 A). The purified enzyme possessed high activity in the elongation assay similar to that of commercial E. coli RNAP (Fig. 5B). In summary, our results demonstrate that HHH samples of the large, multisubunit DNA/RNA-binding proteins (RNAPs) can be obtained in one step within only 5–7 h. Our CL7/Im7 expression/ purification system represents a dramatic (10- to 500-fold) im- provement over the previously used approaches (Fig. 2).
Expression and Purification of Membrane Proteins. Transmembrane proteins constitute up to 40% of the total protein pool in living cells. Most of them are of high functional and clinical signifi- cance, yet only a few have been studied at a high-resolution structural level. This deficit is largely attributable to challenges in expression or purification (30) (SI Appendix, Fig. S5). For our trials, we selected two membrane proteins, one from prokaryotic
and one from eukaryotic organisms. These two proteins differ drastically in size, function, and configuration in the membrane. Bacillus halodurans YidC membrane integrase (molecular mass, ∼32 kDa) is an “all”-membrane protein, without bulky extramembrane domains. The structure of YidC has already been determined (6, 7). It was selected as a reference in our purification. The lengthy original purification protocol (five steps; ∼4 d) (Fig. 2) is one of the most complex used to purify a selected subset of the significant membrane proteins, the crystal structures of which have been determined (SI Appendix, Fig. S5). In particular, the first His-Trap step resulted in only ∼65% purity protein, because other untargeted membrane proteins have substantial binding affinities to the Ni 2 +-activated column (Fig. 6 A). The protocol was also characterized by a rather poor yield (∼1 mg protein from ∼20 g cells). This low yield is likely caused by a low overexpression level typical for membrane proteins and is further compounded by a loss of material during multiple purification steps (Fig. 2). To test our new system, we first adjusted the YidC gene to the E. coli codons and constructed a vector with the PSC-cleavable CL7-tag fused to the C terminus (SI Appendix, Fig. S10A, sche- matic drawing). Upon IPTG induction, the YidC expression level was greatly improved (SI Appendix, Fig. S10A, LYS, Left) com- pared with published studies (6) (Fig. 6A, MF lane). However, almost all YidC remained in the supernatant (SI Appendix, Fig. S10A, SN, Left) upon ultracentrifugation, a traditional first step in membrane protein purification away from cytosolic proteins and NAs (6). One explanation was that, upon lysis, abundant YidC led to very small pieces of membrane fragments, pre- venting them from sedimenting to the bottom of the tube. In- deed, in the absence of IPTG induction, the YidC expression level was reduced, but the membrane fraction increased upon ultracentrifugation (MF; SI Appendix, Fig. S10B). The MF was then loaded onto the Im7 column in a high concentration of NaCl (0.9 M) and detergent (1.5% dodecyl-maltopyranoside; DDM). A high-purity YidC preparation (∼14 mg protein from ∼20 g cells) was obtained in one-step elution with PSC, much higher than the yield in the published work (6). A small (∼ 5 – 7%) but visible impurity was observed in the purified sample (EL; SI Appendix, Fig. S10B). This impurity might be attributable to the covalent linkage of the protein to the membrane components
Fig. 4. Expression, purification, and activity of ttRNAP. (A) Expression levels of ttRNAP using expression vectors developed in this study (Fig. 3). (B and C) A one-step purification of ttRNAP using the His-tag (B) or CL7/Im7 (C) approaches. (D) Transcription elongation assays for the purified, overexpressed ttRNAP enzyme (Left) and the reference WT enzyme isolated from the host without overexpression (Right). 60C, lysate (LYS) heated at 60 °C for ∼45 min; β’ωH8/ β’ωCL7, β’ω fusion with the His8 or CL7 tags; EL, eluate; FT, flow through; LD, molecular mass standards in kilodaltons; RNA18, synthetic 18-mer RNA with fluorescein (FLU) at the 5′-end; T/NT, DNA template/nontemplate strands.
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bacterial MukBEF condensin complex consists of three protein subunits (MukB, MukE, and MukF) and has a molecular mass of ∼540 kDa in a proposed B 2 (E 2 F) 2 configuration (37). An over- expression protocol established for a full E. coli MukBEF complex or its reconstitution from the individually expressed subunits has allowed for protein purification only on a small analytical scale (37). The complex requires metal ions for stabilization and has a ten- dency to dissociate even upon small variations of salt concentrations or under certain chromatographic conditions (37). Accordingly, the crystal structure of only a partial MukBEF complex has been de- termined by using relatively short, truncated subunits (35). We have introduced the PSC-cleavable CL7-tag coding sequence directly to the 3′ end of the Salmonella typhimurium chromosomal mukB gene, which encodes the largest subunit. The derived S. typhimurium was cultivated using a fermenter facility. One milli- gram of the condensin complex was purified from 45 g of cells in essentially the same manner to that of RNAPs, except that PEI precipitation was applied to remove DNA from the lysate in place of the DNase digestion and that a lower (0.5 M NaCl) salt concentra- tion was used during loading. We used a small 1.5-mL Im7 gravity column because of the anticipated low amount of the target complex. The chief purpose of this purification trial was to evaluate the sensitivity of the Im7 column, the target protein purity, the physiological configuration of the condensin complex, and the stoichiometry of its components. Thus, after washing, the bound MukBEF complex was stripped from the column using 6 M Gdn in place of proteolytic elution by PSC (Fig. 7A). This simple procedure yielded 1 mg of high-purity (∼97%) protein free of contaminating DNA (OD 260/280 ratio, ∼0.55–0.65). Stoichi- ometry (BE 2 F) of the complex subunits in the purified sample was as expected (Fig. 7A). Importantly, the identities of the bands were confirmed by mass spectrometry. Unexpectedly, mass spectrometry also identified a prominent band of a molecular
chaperone DnaK, which was present at an equimolar abundance as MukB. DnaK is likely an integral part of the functional con- densin complex, as it resisted dissociation by 0.8 M NaCl applied to the column during purification. There are three major implications from these results. First, the CL7/Im7 approach can efficiently achieve >1,000-fold puri- fication of the large, multisubunit DNA-binding protein complex of very low abundance from the native host in a one-step/1- d protocol. Second, in a striking contrast to the overexpressed/ reconstituted E. coli MukBEF (37), the Salmonella complex was extremely stable, resistant to both high- and low-salt conditions. The difference in stability might reflect intrinsic properties of the condensin complex of different organisms. Alternatively, it could suggest that the overexpressed/reconstituted samples from E. coli may have some folding/assembly defects due to unnatural ex- pression conditions. One distinct possibility is that the over- expression/reconstitution systems lack some essential cellular component(s) required for their proper folding, for complex in- tegrity, or both. The molecular chaperone DnaK is known to assist protein folding and complex assembly. The stoichiometric presence of DnaK in the purified Salmonella MukBEF complex is consistent with its functional role. Finally, only a small amount of the Im7 beads (1.5 mL) were used to obtain 1 mg of purified complex. We there- fore do not anticipate significant difficulties to obtain crystallization quantities (10+ mg) of the protein complex via the same protocol through scaling up the amount of beads and cell lysates.
Expression and Purification of Human RSF1. The human remodeling and spacing factor (RSF) complex consists of two subunits (hSNF2H and RSF1) and was previously implicated in mediating nucleosome assembly (38). RSF1 is 1,441 amino acids in length with a molecular mass of ∼164 kDa. Expression and purification of RSF1 was previously conducted in the insect sf9 cells using the baculovirus system (38). The yield was rather small, limiting in
Fig. 6. Expression and purification of membrane proteins. (A) Expression and a His-Trap purification of B. halodurans membrane integrase, YidC, using a native gene sequence, including the signal peptide (SPN) (6). (B) A one-step YidC Im7 purification following the PEI precipitation. The gene sequence was adjusted to E. coli codon use, and the signal peptide (SP1) has a Cys to Ser mutation. (C) Expression and a one-step Im7 purification of human chaperon CNX. ΔCNX, truncation of the CNX protein; EL, eluate; FT, flow through; LD, molecular mass standards in kilodaltons; LYS, lysate; MF, membrane fraction (pellet fraction after ultracentrifugation); PL, supernatant after PEI pellet was washed with the buffer containing 0.6 M NaCl and 1.5% DDM; SN, supernatant (soluble fraction after PEI). *1 mg of YidC protein in A was obtained after the last (fifth) purification step (see Fig. 2).
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used for pull-down assays, as the Ni 2 +^ beads exhibit intrinsic affinities to heavily charged protein, such as histones. Further- more, a one-step purification via Im7 consistently provided similar quality samples to that of the two-step protocol. In the one-step purification, lysates containing the full-length RSF1 or its largest fragment (F10; Fig. 7B) were directly loaded on the Im7 column in high salt. Thus, a single Im7-based purification protocol could be scaled up for high-yield protein production with no major obstacles. Finally, the CL7/Im7 approach proved to be efficient in pull-down assays, allowing for detailed func- tional characterization of RSF1 and its 12 distinct fragments at the molecular level. We suggest that it could also be effectively used in functional studies of the other biological systems.
Discussion We have developed a CL7/Im7 ultra-high-affinity system, which is capable of a one-step/1-d HHH purification of proteins from the most challenging, distinct “purification” classes (Figs. 4–6). No conventional technique was able to achieve a one-step HHH puri- fication of proteins studied in this work, nor of proteins used for high-throughput crystallographic analysis (4) or other high-impact structural studies (SI Appendix, Figs. S5 and S6). Overall, our HHH purification via the CL7/Im7 system results in a 10–500-fold im- provement in productivity over the previously published, multistep protocols (Fig. 2). A notable illustration of the superior perfor- mance of the CL7/Im7 approach is that the CNX protein sample, which we purified in a few hours and from only a few grams of E. coli cells, would have a market value of ∼$400,000, according to the current commercial prices (SI Appendix, Fig. S11C). Importantly, due to its ultrahigh sensitivity, the CL7/Im7 technique is able to achieve a one-step HHH purification for low-abundance proteins expressed from the context of the chromosomes of the na- tive hosts. This capability is crucial for a big pool of functionally important proteins for which overexpression is not possible. The modern recombineering technologies (39, 40) are capable of genet- ically introducing CL7 tags into any proteins in bacterial or eukaryotic chromosomes, as we have demonstrated with the MukB gene used to purify the MukBEF–DnaK complex (Fig. 7A).
Fig. 7. Purification of condensin complex and RSF1 protein for pull-down assays. (A) A one-step Im7 purification of bacterial CL7-tagged MukBEF condensin complex expressed from the host chromosome in S. typhimurium. (B) Expression and analytical purification of dual (H8/CL7)-tagged human RSF1 and its fragments (FN/CLT), where N is the number of the serial fragments used in pull-down assays. CLT, C-terminal (SM-CL7) tag; SM, SUMO domain. Purification is shown for the full-length protein (RSF1; orange) and some fragments (FN/CLT; brown). *A second (lower) band observed in the Im7-purified samples of all small RSF1 fragments are likely due to proteolytic (by some cellular proteases) excision of the N-terminal, flexible EX1 expression tag, as processing of these fragments with PSC converted the upper band to the lower band. EL, eluate; EX1, expression tag 1 (SI Appendix, Fig. S9B); FT, flow through; Gdn, guanidine hydrochloride; HT, the samples purified using the His-Trap columns were loaded on the Im7 beads and eluted with SDS; LD, molecular mass standards in kilodaltons; LYS, lysate.
vitro functional studies. Due to the large size and unknown characteristics, there has not been an attempt to express and purify RSF1 in prokaryotic cells. To elucidate the function of RSF1, to identify its functional do- main, and to test whether the His-Trap or the CL7/Im7 approach is suitable for purification and functional (pull-down) assays for this eukaryotic protein, we established an expression and purification protocol for RSF1 for E. coli cells. The NA sequence of the RSF1 gene was adjusted to the E. coli codons. We then constructed a dual-tagged (His8/CL7) vector for expressing the full-length RSF1 and 10 serial fragments (Fig. 7 B). In addition, a PSC- cleavable expression tag was introduced to the N terminus. The serial fragments differ substantially in sequence or size from one another. They therefore possess distinct chemical and structural properties and essentially represent unrelated purification targets. RSF1 is likely a multidomain protein, and the exact domain boundaries are difficult to identify based on only the sequence or homology information. Therefore, there is the possibility that some of the arbitrary fragments might harbor structurally in- complete and unfolded domains, affecting their solubility. To ac- commodate for this possibility, we introduced to the C terminus the SUMO domain, which is known to increase solubility of the protein targets (Fig. 7 B). Consistently, no solubility problems were detected for any of the constructs in either the expression or purification trials. In this first experimental design, we used a two-step purification protocol with His-Trap in the first step to evaluate the amount of proteins recovered (Fig. 7 B). A second Im7 step was immediately used in pull-down assays to identify the functionally active frag- ments capable of interacting with proteins in mammalian cells. The minimal functional domain was then further delineated upon additional dissection (Fig. 7 B, fragments F7.1 and F7.2). Our results can be summarized as follows. First, the full-length RSF1 and several fragments purified through the His-Trap col- umn demonstrated significant contaminants (Fig. 7 B). Different fragments demonstrate distinct patterns of impurities, consistent with our prediction that they represent essentially unrelated purification targets (Fig. 7 B). Second, the His-Trap could not be
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