Protein Expression and Purification 93 (2014) 32–37

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Protein Expression and Purification

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Expression, purification, and characterization of full-length bovineleukemia virus Gag protein from bacterial culture

1046-5928/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.pep.2013.10.008

⇑ Corresponding author. Tel.: +1 706 368 5718.E-mail address: dqualley@berry.edu (D.F. Qualley).

1 Abbreviations used: MA, matrix; CA, capsid; NC, nucleocapsid; FIV, felineimmunodeficiency virus; HTLV-1, human T-cell leukemia virus type one; VLPs,virus-like particles; IPTG, isopropyl beta-D-1-thiogalactopyranoside.

Dominic F. Qualley ⇑, Bethany L. BoleratzDepartment of Chemistry, Berry College, Mt. Berry, GA 30149, United States

a r t i c l e i n f o

Article history:Received 2 October 2013and in revised form 10 October 2013Available online 23 October 2013

Keywords:Retrovirus GagSize-exclusion chromatographyViral assemblyBovine leukemia virusMatrixNucleocapsid

a b s t r a c t

In retroviruses, the Gag protein is a precursor from which the mature proteins matrix, capsid, and nucle-ocapsid are derived. Gag plays an important structural role in the assembly of virions at the plasma mem-brane. While Gag proteins from several different retroviruses have been purified for study in vitro, therehas yet to be a report of successful purification of deltaretroviral Gag. In this paper, we report the cloning,expression and purification of full-length bovine leukemia virus (BLV) Gag from Escherichia coli using acombination of polyethyleneimine precipitation, ammonium sulfate precipitation, and affinity chroma-tography. Experiments using size-exclusion chromatography were also performed to analyze the oligo-meric state of the Gag protein in solution, and results suggest that it exists primarily as a monomerbut may oligomerize into higher-order complexes to a small extent. Molecular weight estimation bycomparison of elution volume to a set of protein standards supports the hypothesis that BLV Gag adoptsa slightly extended conformation in solution. The results are discussed in comparison to the solutionstructure and assembly pathways of other retrovirus genera.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Retroviruses are a family of viruses that use a virally-encodedreverse transcriptase to convert their (+)-single stranded RNA gen-omes into double-stranded DNA prior to nuclear import and inte-gration into the host cell’s genome [1]. There are three genescommon to all retroviruses: gag, pol, and env. The gag gene encodesthe structural proteins of the virus; these include but are not lim-ited to matrix (MA),1 capsid (CA), and nucleocapsid (NC). Duringreplication, these proteins are expressed as a single polypeptide,simply referred to as Gag. After assembly and release from the cell,Gag is proteolyzed into its constituent proteins in a process knownas maturation. Prior to release of the immature virion from the cell,the Gag protein plays an important role in viral assembly and gen-ome packaging [2–7]. In human immunodeficiency virus type 1(HIV-1), a well-characterized retrovirus, the NC domain of Gagstrongly binds to the genomic RNA while the MA domain binds tothe plasma membrane of the cell through both electrostatic interac-tions and insertion of a covalently-attached myristoyl group into thelipid bilayer. The CA domain facilitates Gag–Gag interactions, pro-moting multimerization at the inner leaflet of the membrane [8–10].

While MA, CA, and NC have been successfully expressed andpurified as individual proteins from a variety of different retrovi-ruses, expression and purification of full-length Gag has provento be much more challenging. This presents a significant roadblockfor progress in understanding how retroviral assembly works,since pure protein is essential for biophysical studies that examinestructure, binding, and oligomerization. HIV-1 Gag used for in vitroexperiments typically contains several key mutations to preservesolubility and stability. First, the C-terminal peptide (p6) that ex-ists immediately following the NC domain is deleted (commonlyreferred to as a Dp6 mutant). Compared to wild-type HIV-1 Gag,GagDp6 is degraded to a much lesser extent when expressed inbacterial culture [11]. GagDp6 can be purified to 85–90% homoge-neity and with much better yields than the wild-type protein. Sec-ond, two mutations (W316A and M317A) are made to adjacentresidues in the CA domain. This mutant, termed WM-Gag, has beenshown to be predominantly monomeric in solution whereas thewild-type protein is primarily dimeric [12]. A third variant con-tains the Dp6 mutation as well as another deletion, D16–99, whichhas been shown to enhance in vitro assembly of Gag; the purifica-tion of this construct has been reported in great detail [13]. Inter-estingly, full-length Gag from feline immunodeficiency virus (FIV)has been successfully purified using affinity chromatography with-out any modifications, save the 6� His tag [14]. This result is sur-prising, since HIV-1 and FIV are both lentiviruses and their Gagproteins share sequence homology.

D.F. Qualley, B.L. Boleratz / Protein Expression and Purification 93 (2014) 32–37 33

Several Gag proteins from other retrovirus genera have beenpurified from bacterial culture and used for in vitro experiments.Murine leukemia virus (MLV, a gammaretrovirus) Gag was isolatedand its properties in solution were shown to differ significantlyfrom what has been observed for HIV-1 Gag [15]. While evidenceexists that HIV-1 Gag adopts a compact conformation [12,16,17],small-angle X-ray scattering experiments show that MLV Gag hasa rigid, extended structure in solution. Wild-type MLV Gag is alsopredominately monomeric, while HIV-1 Gag exists in a mono-mer–dimer equilibrium. Gag protein from an alpharetrovirus, Roussarcoma virus (RSV) has been purified in its native form with theexception of the C-terminal protease domain, which is normallytranslated as part of the Gag polyprotein in RSV [18]. This led towork showing that RSV Gag required nucleic acid to promote di-mer formation as an intermediate step in assembly [19,20].

To date, successful purification of deltaretroviral Gag has notbeen reported. The most notable human deltaretrovirus is humanT-cell leukemia virus type one (HTLV-1). HTLV-1 is endemic inmany areas of the world, and is the causative agent of adult T-cellleukemia and HTLV-associated myelopathy/tropical spastic para-paresis [21]. Cell culture experiments have shown that, as for otherretroviruses, elements of HTLV-1 Gag are critical for membranebinding and genome packaging [22,23]. Interestingly, there aresome key differences between HIV-1 and HTLV-1 in terms of thebehavior of Gag in solution; HTLV-1 Gag is primarily monomeric,and forms virus-like particles (VLPs) with a poorly organized Gaglattice compared to HIV-1 [24–26].

Bovine leukemia virus (BLV), another deltaretrovirus, infectsdomestic cattle and is prevalent in dairy herds in the United States[27]. Although humans cannot be infected with BLV, infected cattlehave shown reduced milk production compared to healthy animals[28], making study of BLV important from an agricultural stand-point. Additionally, antibodies reactive to BLV capsid have beendiscovered in human blood sera, presumably due to consumptionof products from infected animals [29]. Finally, BLV may also beuseful as an animal model for development of a vaccine forHTLV-1 due to the similarity of BLV and HTLV-1 [30,31]. Previouswork has shown that BLV Gag relies on both its MA and NC do-mains for effective genome packaging; zinc-binding residues inthe NC domain and basic residues in the MA domain appear tobe especially important [32]. Furthermore, myristylation of theN-terminal glycine residue of Gag and the presence of a PPPY motifwere also shown to be crucial for the production of VLPs [33].While the role of the myristate moiety in this context is clear, lessis known about the interaction of BLV Gag with the plasma mem-brane of the host cell. Both MA [34] and NC [35] of BLV have beenpurified and used in biophysical experiments that characterize nu-cleic acid binding, but the use of authentic Gag would provide aclearer and more accurate representation of the events that occurduring replication.

In this paper, we report the first successful purification of a full-length deltaretroviral Gag protein. Cloning, expression, and purifi-cation procedures are described in detail, along with preliminaryexperiments designed to characterize the properties of BLV Gagin solution. Our results show that Gag has been purified to nearhomogeneity, and that it exists in solution as a partially-extendedmonomer.

Materials and methods

Cloning

The DNA sequence corresponding to full-length Gag was ampli-fied by PCR using a plasmid template (pKB426, a kind gift from Dr.Kathleen Boris-Lawrie) which is a derivative of pBLV-SVNEO, a

previously described construct [36]. The forward primer containedan XhoI restriction site at the 50-end of the sequence coding forGag, while the reverse primer contained an AvrII site positionedafter the stop codon at the 30-end. The sequences of the primerswere as follows: 50-GTGACACTCGAGATGGGAAATTCCCCC-30 (for-ward) and 50-GTGACACCTAGGTTAGTTTTTTGATTTGAGGG-30 (re-verse). Both primers were purchased from Integrated DNATechnologies (Coralville, IA) and used without further purification.

Following PCR amplification, the DNA product and the expres-sion vector pET45b (EMD Millipore, Darmstadt, Germany) wereeach digested with XhoI and AvrII (New England Biolabs, Ipswich,MA) using standard molecular biology protocols. The digestionreactions were cleaned up using a PureLink PCR Purification kit(Life Technologies, Grand Island, NY); the High-Cutoff binding buf-fer provided was used to remove DNA less than 300 bp in length.The Gag DNA insert (25 ng) and linearized pET45b (20 ng) wereincubated at 16 �C for 4 h in the presence of 2 Weiss units of T4DNA ligase (EMD Millipore) in a total volume of 10 lL accordingto the manufacturer’s instructions.

The entire ligation reaction was transformed into chemicallycompetent NovaBlue Escherichia coli cells (EMD Millipore). Severalcolonies were screened for the presence of BLV Gag DNA by colonyPCR; colonies that were positive were grown in liquid culture andplasmids were purified using a commercially available miniprepkit (Omega Bio-Tek, Norcross, GA). The presence of BLV Gag DNAwas confirmed by restriction digests of the purified plasmids, andplasmids were sequenced to confirm that the correct sequence hadbeen inserted. The expression plasmid was then transformed intochemically competent Rosetta-2 (DE3) pLysS cells (EMD Millipore),a strain that contains a plasmid encoding a number of tRNA genescorresponding to rare codons.

Protein expression and purification

A small culture (10 mL) of LB media containing 100 lg/mLampicillin and 34 lg/mL chloramphenicol was inoculated with asingle colony of Rosetta-2 (DE3) pLysS cells containing the BLVGag expression plasmid. After shaking overnight at 37 �C, the smallculture was added to 500 mL of LB media (with antibiotics at theconcentrations used for the small culture) and incubated at 37 �Cwith agitation until the optical density of the culture at 600 nmreached 0.5. At this point, the culture was allowed to cool to roomtemperature before addition of isopropyl beta-D-1-thiogalactopy-ranoside (IPTG) to a final concentration of 1 mM. Expression wasallowed to continue overnight at room temperature with shaking,and cultures were harvested by centrifugation for 20 min at8000 rpm. Cell pellets were stored at �80 �C until purification tookplace.

Cell pellets (about 2.4 g wet weight, representing 500 mL of li-quid culture) were thawed on ice and resuspended completely in20 mL of lysis buffer (50 mM HEPES pH 7.0, 0.3 M NaCl, 1 mM tris(2-carboxyethyl) phosphine–HCl, 10% glycerol, 10 mM imidazole,and 1 mM phenylmethylsulfonyl fluoride). The cells were soni-cated on ice using 10 cycles of the following sequence: 1 s on, 1 soff for 20 s; rest on ice for 40 s. Polyethyleneimine was then addedto a final concentration of 0.15%, and the lysate was allowed toincubate on ice for 15 min to precipitate nucleic acids. After incu-bation, a thick white precipitate was observed. The lysate was cen-trifuged at 11,000 rpm for 20 min, and the pellet discarded. To thesupernatant was added 1/2 volume of ammonium sulfate solution(saturated at room temperature), dropwise, on ice, with stirring.Following addition, the solution was allowed to incubate on icefor 30 min without stirring. The solution was then centrifuged at11,000 rpm for 20 min. The supernatant from this step was dis-carded, and the solid white pellet was gently and completely resus-pended on ice with 1 mL of column buffer (50 mM HEPES pH 7.0,

Fig. 1. 1% agarose gel run in TAE buffer showing the successful amplification of theGag gene from the pKB426 template. The approximate size of the PCR product (onthe left) corresponds to the projected size of 1203 bp.

34 D.F. Qualley, B.L. Boleratz / Protein Expression and Purification 93 (2014) 32–37

0.3 M NaCl, 1 mM tris (2-carboxyethyl) phosphine–HCl, and 10%glycerol). The solution was added to a column containing 1 mL ofcOmplete His-Tag Purification Resin (Roche Applied Science, India-napolis, IN) and washed extensively with column buffer containing10 mM imidazole. BLV Gag was eluted using a step gradient ofimidazole in column buffer. Three elution buffers (10 mL each, con-taining 50, 200, and 500 mM imidazole) were added sequentiallyto the column, and the eluate was collected in 5 mL fractions. Frac-tions were run on a 12% SDS–PAGE gel, where the majority of pureGag protein was found to elute at 200 mM imidazole. The Gag-con-taining fractions were pooled and concentrated to a volume ofabout 200 lL using an Amicon centrifugal filtration device with a30 kDa molecular weight cut-off (EMD Millipore); the proteinwas also exchanged into column buffer (without imidazole) at thistime.

The concentration of purified BLV Gag was determined using UVabsorbance at 280 nm with an extinction coefficient of79,800 M�1 cm�1, calculated from the amino acid sequence as de-scribed previously [37]. The measured A260/A280 ratio was about0.5, indicating that the protein was free of contaminating nucleicacids. Zinc acetate was added (2.2 equiv., 1.1 per zinc finger), andthe Gag protein was stored in aliquots at �80 �C until further use.

Size-exclusion chromatography

Size-exclusion experiments were performed using a Yarra SEC-2000 HPLC column (Phenomenex, Torrance, CA) with a length of300 mm and an internal diameter of 4.6 mm. The column was con-nected to a Perkin–Elmer Series 200 HPLC system with the detectorset to 280 nm. For the molecular weight standards and for BLV Gag,a 50 lL sample was injected and eluted at a flow rate of 0.50 mL/min with a buffer containing 50 mM sodium phosphate pH 7.0and 150 mM NaCl. The void volume (V0) of the column was deter-mined by injection of a 1 mg/mL solution of blue dextran whilemonitoring the absorbance at 360 nm. The column was calibratedby sequential injections of 1 mg/mL solutions of the proteins listedin Table 1. A calibration curve was constructed by plotting the ratioof elution volume to void volume (Ve/V0) of each standard vs. logmolecular weight, and an equation for the curve was determinedby linear regression.

Results and discussion

Expression and purification of retroviral Gag from bacterial cul-ture can be a surprisingly difficult endeavor. We initially tried toexpress BLV Gag in two other expression vectors (pET15b andpET32a); however, despite trying a wide variety of expression con-ditions, the protein was only expressed as a truncation product(pET32a) or was not expressed at all (pET15b). Next, we attemptedto express Gag using the vector pET45b, which would produce full-

Table 1Proteins run as standards on Yarra SEC-2000 size-exclusion column. The molecularweights and retention times of the standards were used to construct the calibrationcurve for BLV Gag size estimation (Fig. 3). Blue dextran was used to determine thevoid volume of the column.

Protein Molecular weight (kDa) Elution volume (mL)

Blue dextran 2000 2.08b-Glucosidase 135 2.72Amyloglucosidase 72 2.87Bovine serum albumin 66.5 2.85Ovalbumin 44.3 3.05Horseradish peroxidase 44.0 3.03Myoglobin 17.7 3.41a-Lactalbumin 14.2 3.40Cytochrome c 12.3 3.50

length Gag with a 6� His-tag at the N-terminus. The Gag DNA wasamplified with primers designed to insert an XhoI restriction site atthe 50-end of the sequence and an AvrII site at the 30-end. The sizeof the PCR product corresponded with the expected size (1203nucleotides, Fig. 1).

Following digestion, ligation, and sequencing, the plasmid wastransformed into Rosetta-2 (DE3) pLysS cells for expression. Ini-tially, expression was induced at 37 �C and was allowed to con-

Fig. 2. Samples from Gag purification run on SDS–PAGE (12%). Lane 1 is a proteinstandard (Precision Plus, Bio-Rad, Hercules, CA) with the molecular weight of eachstandard listed in kDa on the left. Lane 2 is the supernatant from the ammoniumsulfate precipitation. Lanes 3–10 represent fractions from the Ni2+ affinitychromatography, consisting of resuspended ammonium sulfate pellet loaded ontothe column (3), eluate from wash step (4), eluate using 50 mM imidazole (5 and 6),eluate using 200 mM imidazole (7 and 8), and eluate using 500 mM imidazole (9and 10).

D.F. Qualley, B.L. Boleratz / Protein Expression and Purification 93 (2014) 32–37 35

tinue for 4 h, with time point being drawn from the culture beforeinduction and every hour post-induction. SDS–PAGE analysis of thesamples indicated that very little protein was being expressed overtime; the protein that was overexpressed was much smaller thanthe expected BLV Gag. The concentration of IPTG used for inductionwas varied from 0.1 to 4 mM with no discernable difference inexpression level. When induction was carried out at room temper-ature and allowed to proceed overnight, there were bands corre-sponding to molecular weights of about 50 kDa (theoretical MWof our Gag construct = 47.7 kDa) and about 16 kDa. When purifica-tion was attempted, the smaller product co-purified with the largerproduct, leading to the conclusion that the smaller protein was a

Fig. 3. Calibration curve of size-exclusion column used to estimate molecularweight of BLV Gag. Identities and molecular weights of the standards are listed inTable 1. The triangle data point, identified with an arrow, indicates the position ofBLV Gag on the calibration curve when its theoretical molecular weight (47.7 kDa)is used.

Fig. 4. Size exclusion chromatography elution profile of BLV Gag when injected as a 5

truncated version of the full-length Gag. Due to this observation,we inserted an ammonium sulfate precipitation step immediatelyprior to column purification. Addition of ammonium sulfate to upto 40% saturation has been used in the purification of other Gagproteins, and was sufficient to precipitate Gag in all reported cases[13,15,18]. For BLV Gag, addition of ammonium sulfate to 33% sat-uration (achieved by addition of 1/2 volume of saturated ammo-nium sulfate solution prepared at room temperature) followed bya brief incubation period on ice adequately precipitated the desiredprotein from solution. After the suspension was centrifuged andthe pellet resuspended in a minimal volume of column buffer,Ni2+-affinity chromatography yielded highly purified BLV Gag(Fig. 2).

The size and shape of BLV Gag in solution was investigatedusing size-exclusion chromatography. Eight protein standardswere run (Table 1) and their elution volumes plotted against thelog molecular weight to generate a standard curve (Fig. 3). BLVGag was injected at a concentration of 5 lM, and the elution profilewas obtained by plotting A280 as a function of time (Fig. 4). Thechromatogram shows a large peak centered around 5.80 min,which corresponds to a molecular weight of about 67.4 kDa. Thisis significantly larger than 47.7 kDa, the calculated molecularweight of purified BLV Gag. Some peak tailing was observed, pos-sibly due to a small amount of conformational heterogeneity ofprotein in the sample. Peak tailing is also often associated withnon-specific interactions between the analyte and stationaryphase, but in this case is unlikely given the high salt concentrationused in the running buffer. A much smaller peak is seen at4.54 min, corresponding to a molecular weight of about 420 kDa.This observation has two potential implications. First, BLV Gag ex-ists predominately as a monomer at concentrations below 5 lM.This is not surprising, given the dissociation constants of other ret-roviral Gag proteins (5 lM for HIV-1 and �400 lM for MLV)[12,15]. Additionally, fluorescence fluctuation spectroscopy exper-iments using HeLa cells expressing HTLV-1 Gag failed to detect anysignificant Gag dimerization in the cytoplasm [25]. Second, theshape of BLV Gag in solution most likely approximates a partiallyextended protein. A protein existing in a rigid, extended conforma-

lM solution. Absorbance at 280 nm was monitored during elution at 0.5 mL/min.

36 D.F. Qualley, B.L. Boleratz / Protein Expression and Purification 93 (2014) 32–37

tion would elute much earlier than expected, as observed for MLVGag. MLV Gag eluted at a volume that corresponds to over twice itscalculated molecular weight, which was explained by small angleX-ray scattering experiments showing that MLV Gag exists in anelongated form [15]. This observation could have potential impli-cations for the packaging of genomic RNA during replication;HIV-1 Gag is much more flexible, and likely adopts a bent confor-mation, allowing both the MA and NC domains to bind the RNA[12,38–45]. Although more evidence is needed to propose thatBLV assembly follows a similar mechanism, purified BLV MA hasbeen shown to bind both viral RNA sequences and inositol hexakis-phosphate, which suggests such a mechanism could be plausible[34]. Although neither the structure of BLV CA nor NC has beensolved, the CA–NC domain of BLV Gag is significantly enriched inPro residues, which could cause the polyprotein to remain inflexi-ble compared to HIV-1 Gag. As determined by NMR, the shape ofBLV MA is globular, unlike that of MLV MA [46].

The identity of the protein eluting in the first peak shown inFig. 4 is much less clear. While it may be tempting to conclude thatthis peak is composed of aggregated BLV Gag, this is unlikely sincethe peak elutes later than the void volume. The molecular weightthat was calculated based on the elution volume (420 kDa) is out-side of the separation range of the column (1–300 kDa); thus,420 kDa can only be considered a rough estimate at best. However,it is interesting that the molecular weight of the early eluting peakcorresponds roughly to that of a BLV Gag hexamer. It has beenshown that retroviruses from different genera (HIV-1, RSV, Ma-son–Pfizer monkey virus, and xenotropic murine leukemia virus-related virus) assemble from hexameric Gag lattices based uponthe N-terminal domain of CA [47–49]. Although the presence ofnucleic acid is required for VLP assembly, it is possible that BLVGag is ‘‘pre-assembling’’ into hexamers prior to RNA-triggeredVLP formation. Our laboratory is currently working on experimentsthat should clarify this issue further.

Acknowledgments

We are grateful to Dr. Kathleen Boris-Lawrie for provision ofpKB426, the National Science Foundation (Major Research Instru-mentation Grant CHE-1125616), and Berry College (Faculty Devel-opment Grant 2013-14-FDG-006). We also thank Drs. KarinMusier-Forsyth, Hally Shaffer, and Alice Suroviec for critical read-ing of the manuscript.

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