Purification of Recombinant Rous Sarcoma Virus IntegrasePossessing Physical and Catalytic Properties Similar toVirion-Derived Integrase
Mark McCord,* Stephen J. Stahl,† Timothy C. Mueser,† C. Craig Hyde,†A. C. Vora,* and D. P. Grandgenett*,1
*Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri 63110; and†National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892
Received November 19, 1997, and in revised form July 20, 1998
The retrovirus integrase (IN)2 is responsible for in-tegration of the linear viral DNA into the host chromo-
some. The viral DNA and IN are packaged within anucleoprotein PIC found in the cytoplasm of newlyvirus-infected cells (1,2). Within the PIC, IN removes adinucleotide from the blunt-ended viral DNA produc-ing 39 OH recessed LTR termini. After transport of thePIC into the nucleus, IN inserts the two viral DNAtermini into cellular DNA in a concerted fashion (full-site integration) producing a small size duplication ofhost sequences (3). The MLV (1,4), HIV-1 (5,6), andRSV (7) PIC have been purified from virus-infectedcells and each can promote the concerted integrationreaction, but with different efficiencies in vitro. One ormore cellular proteins may have auxiliary roles in theintegration pathway (4,5).
Reconstructing the in vivo integration reaction invitro requires purified IN and model retrovirus-likeDNA substrates. IN has been purified from AMV (8,9)and RSV (10) virions, but not from MLV (11). Inte-grases from several different retroviruses have beenexpressed as recombinant proteins (9, 12–15). Previouspreparations of recombinant IN generally retain the 39OH processing activity and have appreciable half-sitestrand transfer activity (the insertion of a single LTRend into a DNA target). However, recombinant inte-grase is generally unable to catalyze the full-site inte-gration reaction efficiently. AMV IN purified from viri-ons can efficiently perform the full-site integrationreaction with linear retrovirus-like DNA substratescontaining LTR sequences at their 39 OH recessed ter-mini (16–18) (Fig. 2).
We have recently reported that purified recombinantRSV PrA IN has the same specific activity as AMV INfor performing the concerted, full-site integration reac-tion (18). In this report, we present the detailed puri-
1 To whom correspondence should be addressed. Fax: (314) 577-8406. E-mail: [email protected].
fication procedure for recombinant PrA IN expressed inbacteria. The protein was purified at high yield to nearhomogeneity. We also investigated the physical char-acteristics of PrA IN such as its solubility propertiesand its subunit structure in relation to the full-siteintegration reaction and compared these data to thosereported for other recombinant integrases.
MATERIALS AND METHODS
Bacterial expression of RSV PrA IN. The RSV INgene was initially isolated from an infectious viralDNA clone derived from the PrA virus strain (19). Astop codon was placed at the appropriate position toproduce IN with the same carboxyl terminus deter-mined for purified AMV IN. The DNA encoding RSV INwas prepared as a NdeI–BamHI fragment using PCR(20) and inserted between these same restriction sitesinto a pET11a vector (Novagen Corporation) to gener-ate an IPTG-inducible construct. The identity of theentire gene was confirmed by DNA sequencing. PrA INwas expressed in Escherichia coli BL21 (DE3) pLysS(Novagen Corporation) grown in a New Brunswick Bio-Flo 3000 fermentor. A single colony of transformedcells was grown in 75 ml of Luria broth with 50 mg/mlof carbenicillin for 12–14 h at 37°C in a shaking incu-bator. This solution was used to inoculate a vesselcontaining 10 L of Terrific Broth (Sigma) with 1% glu-cose (w/v). Cells were grown at 37°C with light aerationat about 2 liters/min air flow and agitation at 300 rpm.When a cell density corresponding to OD600 nm of 1.7was reached, protein expression was induced by addingIPTG to a final concentration of 1.6 mM. After 2 h, thecells were harvested by centrifugation yielding ;45 gof wet cell pellet. The cells were rapidly frozen andstored at 280°C. The expression level was judged bydensitometric scanning of Coomassie blue-stainedSDS–PAGE gels of whole cells collected before andafter induction (Fig. 1).
Purification of PrA IN. The frozen bacterial pastewas thawed on ice for ;30 min in 200 ml of lysis buffercontaining 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 1mM DTT, 1 mM AEBSF (a protease inhibitor), and 1 mMEDTA. All operations were conducted on ice or at 4°C.The presence of T7 lysozyme coexpressed in the cells ledto rapid lysis upon thawing and stirring. Light sonication(Branson Sonifier Model 250, with ½-in. flat probe, usinga 50% duty cycle at 75% power for 1 to 2 min) was used tohomogenize further and partially shear the viscous nu-cleic acid present in the suspension. The homogenizedsamples were subjected to centrifugation in Beck-manTI-45 rotor tubes at 40,000 rpm (;120,000g) for 30min at 4°C. Low-salt (0.1 M NaCl) washes of the pelletwith lysis buffer removed some contaminants but left themajority of IN in the pellet. For the wash procedures, thesuspension was centrifuged as described above. The su-pernatant fraction was discarded and the pellet was sus-
pended in 200 ml of cold lysis buffer using light sonicationand again subjected to high-speed centrifugation. Thepellet was suspended in 200 ml of lysis buffer and thelow-salt wash step was repeated. The pellet fraction fromthis high-speed spin was suspended by light sonication in100 ml of extraction buffer (lysis buffer in which the NaClconcentration was increased to 1.0 M). The suspensionwas allowed to stand on ice for 30 min and then centri-fuged for 15 min at 40,000 rpm. The supernatant fractionwas collected and found by Coomassie-stained SDS–PAGE to contain the majority of IN (Fig. 1).
The supernatant (containing 1.0 M NaCl) showed ahigh absorbency at 260 nm, suggestive of significantcontamination by nucleic acids. These were removed bya “semibatch” cation-exchange column chromatogra-phy step using Pharmacia SP-Sepharose FF (2.6 cmdiameter 3 20 cm bed height, bed volume of about 100ml), preequilibrated with buffer A (50 mM Hepes/NaOH, pH 7.5, 1 mM DTT, 1 mM EDTA, and 10 mMMgSO4). The supernatant fluid from the high-salt ex-traction was diluted slowly with cold water and rapidstirring to reduce the conductivity to ;30 mS, roughlydoubling the volume. The diluted sample was loaded onthe column at a flow rate of 10 ml/min. The column waswashed at 20 ml/min with approximately 300 ml of amixture of 80% (v/v) buffer A and 20% buffer B (sameas buffer A, but with 1.5 M NaCl) until the absorbanceat 280 nm returned to the baseline value. The majorityof the nucleic acid contaminants were eluted in theunbound fractions and discarded. IN was eluted at aflow rate of 10 ml/min using a sharp (1 bed vol), lineargradient from 20 to 100% buffer B and collected in 10ml fractions. Fractions found by SDS–PAGE to containIN were pooled.
FIG. 1. (Left) Coomassie blue-stained SDS–PAGE (Novex Corp.4–20% gradient gels) showing expression levels of PrA IN and prod-uct at various stages of purification. Lane 1, Novex Mark 12 molec-ular weight standards, labeled in kilodaltons; lane 2, whole cellextract of E. coli BL21(DE)pLysS before induction; lane 3, whole cellE. coli extract after a 2-h induction with IPTG (IN is evident as aprominent band migrating between the 31- and 36.5-kDa markers);lane 4, high-salt extract; lane 5, pooled IN fractions from SP-Sepha-rose FF column; lane 6, final purified product following gel filtrationchromatography. (Lanes 1–3, 4, and 5 and 6 were photographed fromdifferent gels). (Right) Coomassie blue-stained 10% SDS–PAGE gelcomparing equivalent quantities (2.5 mg) of purified AMV IN andPrA IN. The small difference in migration distances is likely due tostorage in different buffers.
168 MCCORD ET AL.
A second, higher resolution cation-exchange columnstep (Pharmacia Source 15S) was used to separate INfrom contaminating bacterial proteins. The pooledsamples from the SP-Sepharose FF column were dia-lyzed overnight against 2 liters of 50 mM Hepes/NaOH,pH 7.5, 1 mM DTT, 1 mM AEBSF, 1 mM EDTA, 100mM NaCl, and 10 mM MgSO4 or, alternatively, dilutedwith cold water to reduce the conductivity to 20 mS.Dialyzed or diluted samples containing up to 50 mg ofIN were loaded at 5 ml/min onto a Source 15S column(1.6 cm diameter 3 10 cm bed height, bed volume of 20ml) preequilibrated with 5% buffer B (the same buffersused with SP-Sepharose FF, above). The column waswashed with 2 to 3 bed vol of buffer with 95% buffer A,5% buffer B. IN was eluted with a linear gradient to50% buffer B over 5 bed vol at a flow rate of 5 to 10ml/min and collected in 5-ml fractions. Fractions con-taining IN were concentrated to 5–10 mg/ml usingcentrifugal ultrafiltration devices (Filtron 10K nominalmolecular weight cutoff). The concentrated sample wasdialyzed against storage buffer (buffer S, 25 mMHepes-pH 7.5, 100 mM ammonium sulfate, 0.1 mMAEBSF, 1 mM EDTA, 1 mM DTT, 10% glycerol w/v and10 mM MgSO4). Typically, the protein appeared ho-mogenous as judged by Coomassie-stained SDS–PAGE. Separation of minor lower molecular weightcontaminants appearing in some preparations wasachieved by gel filtration on Pharmacia Superdex 75(26-mm 3 60-cm column). An isocratic elution wasused with a maximum sample load of 50 mg protein ata flow rate of 1 ml/min with buffer S (minus glycerol).Prior to storage, a 1 M zinc sulfate solution was addedto the protein to give a final concentration of 10 mMzinc and kept for one hour at 4°C. Excess zinc sulfatewas removed by buffer exchange into buffer S on aPharmacia fast desalting column (Sephadex G-25 SF,10 mm 3 10 cm). Pooled fractions containing IN wereconcentrated by centrifugal ultrafiltration to 5–10mg/ml and stored at 280°C.
Purification of AMV IN. AMV IN was purified fromvirions to near homogeneity as previously reported(8,10).
Solubility profiling. To determine the solubilityprofile of the recombinant IN, a sample of purifiedprotein was precipitated by dialysis against distilledwater, suspended homogeneously, and dispensed into1.5-ml centrifuge tubes with approximately 0.3 mg pro-tein per tube. The protein was collected by centrifuga-tion at 15,000 rpm in a benchtop microcentrifuge. Thepellets were mixed with 50-ml aliquots of various solu-tions containing either 100 mM buffer or 200 mM saltsand allowed to stand for 5 min at ambient temperatureand then centrifuged as above. Aliquots of the super-natant fraction were diluted into a sample buffer (100mM Tris–HCl, pH 8.0, 100 mM NaCl) and a UV ab-sorption measurement at 280 nm was used to estimate
the amount of solubilized material. Buffers tested in-cluded 100 mM MES/NaOH, pH 5.8; Pipes/NaOH, pH6.5; Hepes/NaOH, pH 7.5; Taps/NaOH, pH 8.5. Saltstested included 200 mM chloride salts (NH4Cl, NaCl,KCl, LiCl, MgCl2, and CaCl2) and a series of 200 mMammonium salts (formate, acetate, cacodylate, pH 7.0;sulfate, phosphate, pH 7.0; and citrate, pH 7.0).
Molar extinction coefficient determination and pro-tein parameters. The theoretical isoelectric point andmolecular weight were calculated using SwissProt Pro-tParam tools available through the internet at http://expasy.hcuge.ch/sprot/proparam.html. The molar ex-tinction coefficient of PrA IN at 280 nm was calculatedfrom the sequence containing 10 Trp and 2 Tyr resi-dues by the method of Gill and Von Hippel (21). Theextinction coefficient was also measured at protein con-centrations of 3.8 and 7.6 mM, both under native con-ditions and while denature in 6 M Gnd–HCl.
N-terminal amino acid sequencing. Purified PrA INwas separated by SDS–PAGE and transferred to aPVDF membrane. The blotted sample was subjected to10 cycles of automated N-terminal Edman degradationusing an Applied Biosystems Model 477A protein se-quencer.
Mass spectrometry. MALDI spectra were obtainedon a Kratos MALDI III instrument operated at anaccelerating voltage of 22 KV. The sample was dis-solved in 0.1% TFA and 50% acetonitrile and applied tothe target in an a-cyanocinnamic matrix.
Protein–protein crosslinking analysis. A total of 1.3mg of IN (50 nM in a 400-ml volume) was preincubatedunder full-site strand transfer assay conditions in theabsence of DNA (18). DSG dissolved in dioxane (4 mg/ml) was added to a final concentration of 2.5 mM andimmediately incubated at 37°C for 20 min. The reac-tions were stopped with the addition of 1 M Tris–HCl(pH 7.5) to a final concentration of 50 mM. To removethe PEG, the crosslinked samples were diluted seven-fold with 50 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1mM EDTA and centrifuged in Millipore Ultrafree-15filtration units (MWCO 5 30,000). The samples werewashed four more times. The retained sample volume(;60 ml) was treated with a 53 concentrated SDS–PAGE dye mixture containing 50 mM Hepes (pH 7.5).After heat treatment, the samples were subjected toSDS–PAGE. The proteins were transferred to PVDFmembranes and subjected to Western blot analysisusing rabbit antiserum directed against the full-lengthPrA IN (19). A specific carboxyl-terminal antiserum toPrA IN was also used. Rabbit antiserum directedagainst a peptide that contained the last 10 carboxyl-terminal amino acids of IN demonstrated that the car-boxyl-terminus of PrA IN remained intact (19). Theproteins were illuminated with ECL (Amersham) andexposed to X-ray film.
LTR DNA donor constructs. Double-stranded oligo-
169RECOMBINANT ROUS SARCOMA VIRUS INTEGRASE
nucleotides (60 bp) containing terminal U3 and U5LTR sequences were cloned into the NdeI site of plas-mid pUC19 that lacked its HindIII site. The supF genewas inserted into the HindIII site located in the middleof the oligonucleotides to generate a 480-bp donor mol-ecule, designated wt U3/U5 LTR donor (18). Donorscontaining mutations in the U3 and U5 LTR terminalsequences were constructed in a similar fashion. Plas-mids were digested with NdeI, and the appropriateLTR donor fragments were isolated by agarose gel elec-trophoresis. A similar size donor (M-2) containing wtLTR termini was previously described (16, 17). Thetarget DNA was supercoiled pGEM.
Labeling of donor fragments and assay for strandtransfer. The donor fragments were 59-end labeled byT4 polynucleotide kinase using [g-32P]ATP (16). Thespecific activities of the donor fragments were approx-imately 6000 cpm/ng DNA.
Reaction mixtures (20 ml) for full-site strand transfercontained 20 mM Hepes buffer (pH 7.5), 1 mM DTT, 8%PEG, 15% dioxane, 5 mM MgCl2, and 330 mM NaCl(18). PrA IN or AMV IN were first preincubated on icefor 10 min in the above assay mixture with 15 ng ofdonor DNA. With 50 nM IN, there are estimated to be12 IN dimers per donor end. The strand transfer reac-tion was initiated by the addition of target (100 ng) andimmediate incubation at 37°C for 10 min. The reac-tions were terminated by the addition of SDS andprotease K to a final concentration of 1% and 1 mg/ml,respectively. The DNA products were separated onagarose gels. The quantity of donor–target recombi-nant products that were produced was determined byMolecular Dynamics PhosphorImager measurements.
The strand transfer assay is based on the ability ofIN to insert linear 480-bp LTR donor fragments into acircular DNA target (16). The integration reactionsinvolve either a single LTR end insertion (half-site) orthe concerted insertion of two LTR termini from indi-vidual donors (bimolecular) into pGEM (Fig. 2). BglIIdigestion of the half-site and full-site donor–target re-combinants produces characteristic labeled donor–tar-get restriction patterns that differentiate termini us-age by IN for strand transfer (16–18).
RESULTS
Purification of RSV PrA IN. PrA IN has been ex-pressed in bacteria and purified to near homogeneity.Steps in the purification are summarized in Table 1and the purity of the product at various stages is shownin the gels of Fig. 1. The recombinant IN is foundassociated with the “insoluble” pellet fraction fromlysed cells, but can be obtained in soluble form in highyield (without detergents) by using a series of low-saltwashings, followed by high-salt extraction. Contami-nating nucleic acids are removed by a semibatchmethod using SP-Sepharose FF and IN is separatedfrom other proteins using high-resolution ion-exchangeand gel-filtration methods. The overall yield of IN isestimated to be as high as 60% and the preparation isapparently homogeneous, or nearly so, as determinedby SDS–PAGE.
A variety of salts and buffers was tested for theireffects on IN solubility. The results of these simpletests indicated that IN is more soluble at lower pH andvery soluble in the presence of MgCl2 and (NH4)2SO4.IN dialyzed in a final buffer system of 100 mM Na–Pipes, pH 6.5, 50 mM NH4Cl, and 10 mM MgSO4 canbe concentrated to above 40 mg/ml using centrifugalultrafiltration.
Amino-terminal sequencing and mass-spectroscopicexamination of the final product further confirmed itsauthenticity. Ten cycles of automated Edman degrada-tion provided the sequence “PLREAKDLHT” corre-sponding precisely to the predicted sequence (19).Within the limit of detection, the initiator Met residuewas quantitatively removed by bacterial processing.The protein mass measured by mass spectroscopy was31,715 6 100 kDa, in good agreement with the mass of31,735 kDa calculated from the sequence. The mea-sured extinction coefficients e280 nm of native and de-
TABLE 1
Purification of RSV PrA IN Expressed in E. coli
Step%
Yielda%
Purityb Lanec
Cell lysis and sonication 100 15 3Low-salt rinses .95 20 —High-salt extraction .90 25 4Bulk fractionation of SP–Sepharose FF .80 25–30 5High-resolution cation-exchange, Source S .60 .95 —Preparative gel filtration on Superdex 75 .60 .95 6
Note. Steps in the purification of bacterially expressed RSV PrAIN. Assays of enzymatic activity were performed only with the final,highly purified samples.
a The yield of IN was estimated by densitometric scanning of gels.b Purity was estimated roughly as the IN band stain density ex-
pressed as a percentage of the total stain density of all proteinspecies.
c See lanes of Fig. 1 for representative appearance of proteins ineach sample following these steps.
FIG. 2. Schematic for full-site integration of retrovirus-like DNAdonors into a circular target. Two 480-bp donors (bimolecular reac-tion) containing 39 OH recessed LTR termini are inserted in a con-certed fashion by IN into pGEM producing a linear 3.8-kb product. Ahalf-site reaction involves the insertion of a single LTR terminus byIN into pGEM producing a circular product with an extended tail.
170 MCCORD ET AL.
natured RSV IN averaged 59,300 M21 cm21 (65%),comparing favorably to the calculated value of 59,940M21 cm21. The concentration of pure protein was de-fined using the absorbance at 280 nm where one OD280 nmcorresponds to a concentration of 1.87 mg/ml.
Physical properties of PrA IN and AMV IN. Weanalyzed the physical state of PrA IN at low and inter-mediate nanomolar concentrations by protein–proteincrosslinking and glycerol gradient sedimentation to de-termine whether it exists as a dimer as had previouslybeen shown for AMV IN (8).
The subunit structure of PrA IN and AMV IN wasinvestigated by glycerol gradient sedimentation at in-termediate protein concentrations (300 to 1000 nM).The 5 to 20% glycerol gradient buffer composition wasidentical to full-site strand transfer assay conditionswith PEG omitted due to possible interference withsedimentation. Both PrA IN and AMV IN sedimentedas dimers at initial layering concentrations between1000 nM (Figs. 3A and 3B, respectively) and 300 nM(data not shown) (8). Half-site and full-site strandtransfer activity comigrated with IN as analyzed bySDS–PAGE (data not shown) and cosedimented withmonomeric BSA (66 kDa). Similar results were ob-tained with PrA IN during glycerol gradient sedimen-tation when 0.1 M ammonium sulfate dialysis bufferwas used instead of NaCl, either with or without BSA(data not shown). The results demonstrate that all ofthe detectable PrA IN sedimented as a dimer at initiallayering concentrations of 1000 nM or less.
Protein crosslinking of PrA IN and AMV IN at 50 nMconcentrations in normal full-site strand transfer con-ditions, in the presence of PEG and absence of DNA,confirmed that both proteins were dimeric, but couldalso form tetramers (Fig. 4A). The crosslinking wasconducted for 20 min at 37°C. Crosslinking of IN for120 min at 4°C, or 60 min at 20°C, also yielded thesame crosslinked species (data not shown). Titration ofPrA IN from 6 to 50 nM, with or without PEG present,demonstrated that the majority of IN was able to formdimers with minor populations of trimers and tetram-ers (Fig. 4B). The efficiency of crosslinking PrA INmonomers to form dimers appeared to be better with-out PEG at 50 nM (Fig. 4B, lanes 4 and 8). Recovery ofprotein and possibly the formation of higher order mul-timers were slightly better with PEG. Similar proteintitration results were obtained when AMV IN wascrosslinked (data not shown).
In summary, both avian IN proteins form dimers atlow and intermediate nanomolar concentrations.
Full-site and half-site strand transfer activities ofPrA IN. We tested the ability of PrA IN at 50 nM orlower to promote the full-site and half-site strandtransfer reactions in comparison to AMV IN. Otherreports of in vitro strand transfer assays have usedrecombinant IN at 100 to 300 nM or higher concentra-tions and for extended incubation times at 37°C.
PrA IN at low concentrations catalyzes both the half-site and the full-site strand transfer reactions with thewt M-2 donor in an equivalent fashion to AMV IN with
FIG. 3. PrA IN and AMV IN sediment as dimers upon glycerol gradient sedimentation. PrA IN (A) and AMV IN (B) were cosedimented withBSA in 5 to 15% glycerol gradients. Sedimentation was from right to left. The buffer contained all of the reagents used for full-site strandtransfer except for PEG. BSA was used as a cosedimentation marker in the same tube containing IN. Aliquots were taken for reading BSAconcentrations (F) at either 280 or 590 nm using a Bio-Rad protein assay method, SDS–PAGE (data not shown), and IN strand transferactivity (10 min at 37°C). The percentage of input M-2 donor incorporated into pGEM was determined. The initial concentration of bothproteins prior to sedimentation was 1000 nM. Recovery of strand transfer activities was estimated to be ;50% in both cases.
171RECOMBINANT ROUS SARCOMA VIRUS INTEGRASE
330 mM NaCl (Fig. 5). At 50 nM IN, ;15 and ;4% ofthe donor was incorporated into pGEM as full-site andhalf-site products, respectively (17,18). Increasing theconcentration (;60 nM or higher) of PrA IN in thestandard reaction mixture (donor DNA was held con-stant) causes a loss of strand transfer activities similarto that observed with AMV IN (17). When both proteinsare above ;60 nM, the donor–donor and the half-site
donor–target reactions are favored over the bimolecu-lar donor–target full-site reaction. At higher concentra-tions of PrA IN (200 nM), the total level of all strandtransfer activities was ;10% of that observed at 50nM. This loss of IN full-site and half-site strand trans-fer activities was still observed with both proteins evenif the standard IN–donor end ratio (12 to 1) is main-tained, but the inhibition was less dramatic. The re-sults suggest that inappropriate protein–protein orprotein–DNA interactions, those incapable of partici-pating in the formation of nucleoprotein complexesthat promote full-site integration, predominate at thehigher protein concentrations.
The solubility of PrA IN is best in the 0.1 M ammo-nium sulfate dialysis buffer in the absence of monova-lent cations. We investigated whether replacing NaClwith ammonium sulfate would improve strand transferactivity. Substitution of ammonium sulfate (50 to 200mM) for 330 mM NaCl (Figs. 5 and 6) with either PrAIN or AMV IN resulted in efficient half-site donor–donor and donor–target reactions but not the full-sitereaction (data not shown). For example, with 0.1 Mammonium sulfate and 50 nM IN (donor DNA was heldconstant with increasing concentrations of IN), ;1% ofM-2 was incorporated as full-site products with ;40%of the input M-2 incorporated into half-site integrationproducts in 10 min. No significant increase or decreaseof strand transfer activity was observed in the presenceof 0.1 M ammonium sulfate between 50 and 200 nM IN.These data suggest that IN can efficiently promoteprotein–DNA interactions in the presence of ammo-nium sulfate for half-site catalysis but not the protein–protein interactions between two IN/LTR donor com-plexes for full-site integration.
FIG. 4. Chemical protein crosslinking of PrA IN and AMV IN. (A). Both proteins at 50 nM were crosslinked with DSG and subjected toSDS–PAGE gel electrophoresis as described under Materials and Methods. The proteins were transferred to PVDF membranes and subjectedto Western blot analysis with rabbit antiserum directed against PrA IN. The lane numbers are indicated at the top. Molecular weightmarkers (not shown) and the 94-kDa b polypeptide of the avian reverse transcriptase (lane 1) established the size of the IN multimers. Theabbreviations in the middle are monomer (M), dimer (D), and tetramer (T). (B). A constant amount of PrA IN (1.3 mg) was incubated underassay conditions without DNA at IN concentrations (see below) as indicated in lanes 1 to 4 with PEG or lanes 5 to 8 without PEG. The volumeof the reaction mixture varied from 3.2 ml for 6 nM IN to 0.4 ml for 50 nM IN. As with IN, a constant amount of DSG (2.5 mM at 400 ml) wasadded to each tube. The samples were analyzed as described in A.
FIG. 5. Comparison of PrA IN to AMV IN full-site and half-sitestrand transfer activities at low nanomolar concentrations. Standardstrand transfer conditions were used to measure activities of bothproteins at 37°C for 10 min. Equal aliquot of the reactions wassubjected to 1% agarose gel electrophoresis. The gel was dried andexposed to X-ray film. Lanes 1, 3, 5, and 7, AMV IN at 6, 12, 25, and50 nM, respectively. The even-numbered lanes are PrA IN at equiv-alent protein concentrations. The concentration of the M-2 donor washeld constant throughout. At 50 nM IN, the molar ratio of IN dimersto donor ends is 12 to 1, respectively. The full-site and half-sitedonor-target recombinant products and the unincorporated M-2 do-nor are indicated on the right.
172 MCCORD ET AL.
Recognition of wt and mutant LTR termini for full-site catalysis. The ability of several IN proteins torecognize wt and mutant LTR termini similarly forhalf-site and full-site catalysis would suggest that theability of each protein to form the active complexes thatpromote these reactions must also be similar. We in-vestigated these phenomena using both wt and mutantLTR substrates.
The efficiency of the half-site reactions is generallybest if the concentration of salt (usually NaCl) is at;100 mM or less. PrA IN can catalyze the half-sitereaction at 100 mM NaCl (Fig. 6B, lane 3). The donor isU5P5,6-TT/AA that has a gain-of-function mutation inthe U5 LTR terminus and a wt U3 LTR terminus (18).The wt U5 end sequence is 59-GCTTCAOH, the mutantU5 sequence is 59-GCAACAOH, and the wt U3 terminusis 59-ACTACAOH. Both AMV IN (Fig. 6A) and PrA IN(Fig. 6B) can catalyze the full-site reaction when theNaCl concentration in the reaction mixture is in-creased to between 300 and 400 mM. Similar data wereobtained with PrA IN using the wt U3/U5 LTR donorupon increasing the concentration of NaCl to 350 mM(data not shown). The results suggest that PrA INinteractions with the LTR termini to form a protein–DNA complex for the half-site reaction are similar toAMV IN. In the same fashion, the two proteins respondsimilarly to increasing NaCl concentrations in com-pleting the full-site reaction.
PrA IN also has the same response to DMSO anddioxane in the presence of varying concentrations of
NaCl for full-site catalysis (data not shown) as seenpreviously with AMV IN (17).
In summary, PrA IN and AMV IN appear to havenearly identical strand transfer capabilities under var-ious assay conditions using either wt or mutant LTRtermini.
DISCUSSION
The initial steps of the purification strategy weredeveloped from leads provided by Marczinovitis et al.(22) who showed that bacterially expressed, geneticallyaltered RSV IN apparently is not soluble and remainsin the pellet material after centrifugation of disruptedbacteria. The majority of IN was isolated by successivewashings of the pellet with 0.1 M NaCl and detergentsand then extraction with buffered 1 M NaCl. Since theprotein purified by the protocol presented here wasintended for crystallization studies, detergents wereeliminated because of potential interference with crys-tallization. However, the yield of extracted IN with 1 MNaCl, even in the absence of detergents, was quitehigh, estimated at greater than 90% of the total ex-pressed IN (Table 1). We found that care must be takento avoid excessive sonication of the pellet fractions inthe early stages of the preparation as this can causelower yields of IN. More thorough homogenization withits greater shearing of DNA evidently releases smallerprotein/nucleic acid complexes that do not sedimenteven in the high-speed centrifugation steps during thewashing of the pellet.
FIG. 6. Effect of NaCl on full-site and half-site strand transfer activities of AMV IN and PrA IN. AMV IN and PrA IN are in A and B,respectively. Standard assay conditions with the 480-bp mutant LTR donor (U5P5,6-TT/AA) were used except the concentration of NaCl inthe preincubation mixture was changed as indicated by the wedge. No IN was present in lane 1 of both sets. The salt concentrations in lanes2 to 6 in A and B were 50, 100, 200, 300, and 400 mM, respectively. Lane 7 of PrA, 500 mM NaCl. The concentrations of AMV IN and PrAIN were at 70 and 50 nM, respectively. The half-site and full-site donor–target products, the donor–donor product, and unincorporated donorare indicated in the middle.
173RECOMBINANT ROUS SARCOMA VIRUS INTEGRASE
The extracted IN following high-salt extraction iscontaminated with nucleic acids that appear to reducethe solubility of the protein and affect its reproduciblebinding behavior on a variety of chromatographic ma-trices. Conceptually, this method of separation of thenucleic acids requires finding the salt concentration atwhich nucleic acids are released from IN and washedaway while IN is retained on the column. IN is stronglybasic (calculated pI of about 10.5) and binds tightly toa cation exchanger even in the presence of relativelyhigh salt. By carefully diluting the high-salt extract toa conductivity of about 30 mS, IN will release its boundnucleic acid as it binds to the cation exchanger. Caremust be taken also when diluting the high-salt extractas rapid dilution with water, or dialysis against a so-lution with low ionic strength, can cause precipitationof the protein, presumably due to reassociation withthe nucleic acid present. A hydroxyapatite column wasalso found to bind IN in the presence of relatively highNaCl concentrations and to separate the protein fromnucleic acid using an increasing ammonium sulfateconcentration gradient (data not shown). However, thecation-exchange step using SP-Sepharose yielded afaster and more nearly quantitative recovery of INusing a sharp gradient of buffered NaCl. Once thenucleic acids are removed, the protein exhibits im-proved solubility and is easy to handle through a num-ber of conventional chromatographic steps, such as thehigh resolution Source S cation-exchange and gel-fil-tration steps reported above.
Because no special effort was made to supplementthe fermentation medium with zinc salts, the finalpurified enzyme was treated briefly with a zinc solu-tion. A small increase in enzymatic activity observedfollowing treatment (data not shown) is presumablydue to saturation of the zinc-binding motif of the N-terminal domain of IN. The extent of Zn21 binding mayexplain in part differences in the observed properties ofthe purified integrases. With recombinant HIV-1 IN,Zn21 promotes multimerization of monomers to dimersand dimers to tetramers or to higher multimers (23,24). The N-terminal zinc-binding domain (residues 1 to55) of HIV-1 IN is apparently responsible for thedimerization of this segment of IN (25). The possiblestructural role that Zn21 has in the formation of PrAIN dimers or higher order multimers is under investi-gation.
One major goal of our recombinant expression of INin bacteria was to provide the quantities of proteinneeded for structural studies, such as X-ray crystallog-raphy. Crystallization of proteins is often critically de-pendent on having highly concentrated material of thehighest purity. Other methods for the purification ofretroviral integrases reported in the literature wereconsidered in the development of this procedure. Bush-man and Wang (26) made use of histidine tags for therapid purification of RSV PrC IN; however, they were
not exploited here because of the likely interference ofthe affinity ligand in crystallization. Subsequent re-moval of affinity tags or fused protein domains involvesextra steps and the use of expensive reagents andgenerally is not amenable to larger-scale preparations.For these reasons, we felt that to express only theauthentic protein and to work out an efficient purifica-tion method for it would prove ultimately to be themost effective approach. Again, because we were inter-ested in crystallizing these proteins, we took the con-servative approach of completely avoiding use of deter-gents during the purification on the chance thatresidual detergents might also interfere with crystalli-zation or cause problems with reproducibility.
As mentioned above, the poor initial solubility of thisprotein seems to be largely due to the presence ofbound nucleic acids. Earlier reports (27–29) describingthe purification of RSV SRB IN involved a high-salt/PEG/dextran method in the early stages of purifica-tion. No doubt this step has the same effect of separat-ing IN from nucleic acids. However, in our hands, thismethod has been difficult to reproduce and yields havebeen unreliable. Asante-Appiah et al. have recentlyreported a method by which retroviral IN (particularlyHIV-1 IN) can be isolated from the bacterial pellet inthe presence of 1 M salt using an immobilized metalion (nickel) affinity column (30). This procedure report-edly makes use of the enzyme’s innate metal-bindingproperties evidently to capture the enzyme to the col-umn under high-salt conditions in which electrostaticinteractions binding nucleic acids are broken.
We had tested purification of RSV PrA IN also to animmobilized metal ion-affinity column (data notshown). This step was employed in the later stages ofpurification (rather than in an early capture mode asabove) as an attempt to remove minor protein contam-inants. Although the protein bound well and could bereadily eluted with an increase imidazole gradient, wefound no increase in purity. We were also concernedabout the required elimination of reducing agents suchas DTT during this step and the possible stripping ofthe zinc-binding domain during elution with chelatingagents and possible contamination with nickel. We alsofound that IN binds to a zinc-charged column, butagain, purity was not increased. The solubility of theenzyme appeared better if reducing reagents were keptpresent throughout the preparation. While the utilityof a nickel column in the early stages of purificationhas been demonstrated (30), the approach used hereeliminates several of these concerns that could affectthe preservation of structural integrity and activity.
The solubility screens were done in order to find theoptimal buffer species, pH, and salt additives to effectgreater solubility. This information is often needed inscreening for protein crystallization before which onemust generally try to attain relatively high proteinconcentrations, typically at least 10 mg/ml. The ammo-
174 MCCORD ET AL.
nium sulfate and MgSO4 additives used in the storageof the protein were found in this way.
A protein’s ability to crystallize has historically beentaken as the final criterion of its purity. To date, therehave evidently been no reports of the crystallization ofa similar full-length retroviral integrase. RSV PrA INpurified according to this protocol has produced smallcubic-shaped crystals using ammonium sulfate as aprecipitant. However, the crystals are too small and toopoorly formed for diffraction use. The crystal qualityproblem seems more likely due to intrinsic propertiesof this protein, rather than to its purity.
We wanted to address several issues in these exper-iments to understand how recombinant PrA IN has thecapacity to efficiently promote the full-site integrationreaction, in comparison to AMV IN. Using the purifi-cation protocol presented in this report, the PrA IN wehave isolated appears to have similar physical andcatalytic properties to the virion-derived AMV IN. Bothproteins exist minimally in a dimeric state under lownanomolar protein concentrations and appear to havesimilar although not identical solubility properties.The sedimentation, protein crosslinking, and catalysisstudies (Figs. 3–6) suggest that recombinant PrA INhave the protein structural features necessary to pro-mote efficient full-site integration.
Avian retrovirus IN have been expressed and pu-rified using cloned SRB (27,28) and PrC (26) strainsof RSV. The deduced amino acid sequence of PrA(19), PrC (31), and SRB (32) IN are nearly identicaland probably very similar to the IN gene of the majorMAV-1 leukosis helper virus population found inAMV virion preparations (8). The mechanisms allow-ing AMV IN and recombinant PrA IN prepared asdescribed above to promote the full-site integrationreaction to a significantly better degree than SRB INis unknown, but it is not likely due to the primarysequence of avian IN. SRB IN can faithfully promotethe full-site integration reaction to produce the avian6-bp host site duplication as analyzed using a 300-bpblunt-ended LTR donor substrate (33). However, theefficiency of the reaction by SRB IN is very low (0.5%of donor substrate is incorporated in 90 min) andrequires HMG1, a nuclear DNA-bending protein(34), to achieve this level of strand transfer activity(33). We have not investigated how effective PrA INis in promoting the full-site reaction using blunt-ended LTR donor substrates, but it may be as inef-fective as SRB IN (33) and AMV IN (17). SRB IN andPrC IN also require relatively higher concentrationsof IN (350 to 3000 nM range) to effectively catalyzethe 39 OH trimming reaction or half-site strandtransfer reactions using double-stranded oligonucle-otides containing LTR DNA terminal sequences(26,29,35). In summary, it is unlikely that differ-ences in the full-site catalysis efficiencies are due toprimary sequences in IN.
The ability of this preparation of recombinant PrAIN to promote the full-site integration reaction may bedue to its formation of stable dimers at low nanomolarconcentrations. The native form of the avian IN proteinis a dimer (8) and is produced in the virion by proteo-lytic cleavage of the dimeric parental molecule, the bsubunit of the reverse transcriptase (36). In contrast topurified PrA IN or AMV IN (Figs. 3 and 4), SRB INrequires concentrations in the 350 to 3500 nM range toproduce a majority population of dimers, as analyzedby protein crosslinking (37) and sedimentation equilib-rium experiments (29). The Kd (monomer-dimer) forSRB IN is 1 to 5 mM (29). The reason for the apparentability of PrA IN to form stable dimers at lower proteinconcentrations relative to other avian recombinant INis unknown but may be related to differences in theexpression and purification procedures used for theseproteins as discussed earlier.
There are some minor physical and catalytic differ-ences between AMV IN and PrA IN. First, with assayconditions (NaCl as monovalent ion) to test for strandtransfer and 39 OH processing, PrA IN loses activityslightly faster than AMV IN upon increasing the con-centration of IN above ;60 nM for strand transfer and;20 nM for 39 OH processing (18). At high concentra-tions (.5 mg/ml) of PrA IN in the absence of assaybuffer conditions, IN prefers the presence of 0.1 Mammonium sulfate with 10 mM MgSO4 instead ofmonovalent cations for solubility (see above). Magne-sium sulfate was routinely present in all of the purifi-cation steps for PrA IN. Divalent metal ions are criticalfor catalysis and possibly for the assembly of IN–DNAcomplexes for catalysis (14, 15, 38, 39). It is unknown ifAMV IN prefers ammonium sulfate and MgSO4 ionsfor solubility at these high protein concentrations.AMV IN is routinely soluble at ;1 mg/ml in a 0.5 to 1M NaCl buffer system (8).
The three main chemical components that affect thefull-site strand transfer reaction appear to be NaCl,dioxane, and PEG, in addition to Mg21 for catalysis(16,17). Salt concentrations in the 350 mM range areessential for both proteins to promote full-site catalysis(Fig. 6). The formation of IN–DNA complexes for half-site and full-site strand transfer parallel each otherupon increasing the concentration of IN to ;60 nM(Fig. 5) (17). Recently, recombinant MLV IN has beenshown to faithfully promote the concerted integrationreaction with double-stranded oligonucleotides in thepresence of 0.4 M NaCl (40). The fidelity of the MLV-specific 4-bp host site duplication is dependent uponhigher salt concentrations. The above results suggestthat the conformation of IN bound to the LTR terminusis modified in the presence of high salt allowing twoindependent IN–LTR donor complexes to associate inan appropriate pairwise manner for the full-site reac-tion. The ability to study these reactions in more detail
175RECOMBINANT ROUS SARCOMA VIRUS INTEGRASE
with full-length, native IN protein from other sourcesmay now be possible.
ACKNOWLEDGMENTS
This work was supported in part by NCI Grant CA16312. Wethank P. Spinella (NIAMS/PEL) for help with N-terminal peptidesequencing, Drs. P. Lecchi and L. K. Pannel (NIDDK) for help withprotein mass spectrometry, W. Idler for help with bacterial fermen-tation, and Dr. Joan Hanley-Hyde for critical reading of the manu-script.
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