Vol. 1, 429-437, September 1990 Cell Growth & Differentiation 429

Expression of the Human Retinoblastoma Gene Productpp1 10RB in Insect Cells Using the Baculovirus System’

Nan Ping Wang, Yuewei Qian, Albert E. Chung,Wen-Hwa Lee, and Eva Y-H. P. Lee2Department of Pathobogy-0612 and Center for Molecular Genetics,University of California at San Diego, La Jolla, California 92093[N. P. W., Y. Q., W-H. L., E. V-H. P. L.], and Department of BiologicalSciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

[A.E.C.]

AbstractThe product of the retinoblastoma susceptibility gene(RB) was overproduced in cultured insect cells usingthe baculovirus expression system. Upon insertion ofthe cloned human RB complementary DNA sequenceinto the viral genome downstream of the promoter ofthe polyhedrin gene, full-length RB protein with an ap-parent molecular weight of 1 10,000 was expressed inthe insect cells. This protein was found to be phosphor-ylated, located in the nuclei of the infected cells, andimmunologically indistinguishable from ppl 1ORB of hu-man cells as assayed by several anti-RB antibodies. FoI-lowing cell disruption and a one-step immunoaffinitychromatographic purification, 6-12 mg of solubleppl iO’� with approximately 95% purity were obtainedper liter of infected suspension culture. Characteriza-tion of the two known biochemical properties of RBprotein showed that this purified protein from insectcells behaved similarly to the authentic human ppl 10KB#{149}

First, it bound to DNA, and second, it could form aspecific complex with SV4O T antigen in vitro. Prompttranslocation of the protein from cytoplasm to nucleusafter microinjection further indicated that the purifiedRB protein may be active. The availability of soluble,intact, and presumably active ppl 1ORS in large quantityrepresents a significant advance for studying the bio-chemical and biophysical properties of the RB geneproduct as well as its potential biological function incancer suppression.

IntroductionRetinoblastoma, a rare childhood cancer of the devel-oping retina, is the prototypic model for studies of reces-sive oncogenesis. Based on the localization of the in-volved genetic element to chromosome i 3q i 4 (i -3) andevidence of its recessive nature (4-6), the putative cancersuppressor gene, retinoblastoma susceptibility gene (RB),was cloned (7-9). This gene contains 27 exons dispersed

within 200 kilobases of genomic DNA and expresses a4.7-kilobase mRNA transcript in all normal tissues ex-amined (10-12). Sequence analysis of the complemen-tary DNA clones revealed a long open reading frame thatcould encode a hypothetical protein of 928 amino acids(9). Using antibodies raised against selected epitopespredicted from the RB cDNA3 sequence, the RB geneproduct has been identified as a nuclear phosphoproteinwith a relative molecular mass of 110,000-i i4,000, andhas been named ppii0RB (13). In addition to retinoblas-toma, the loss of RB gene function has also been impli-cated in the development of several other tumor types,including breast cancer (14, i 5), osteosarcoma (1 6, 1 7),prostate cancer (18), and small cell lung carcinoma (19-2i). The recent demonstration that the reintroduction ofRB gene, via retrovirus-mediated gene transfer, into reti-noblastoma, osteosarcoma, or prostate carcinoma cellsapparently suppresses several aspects of their neoplasticphenotype, including tumorigenicity in nude mice (i8,22), provides direct evidence for the tumor suppressionfunction of the RB gene. However, the molecular basisof this biological activity has not been defined to date.

Elucidation of the biochemical properties and biolog-ical functions of the RB gene product has been hamperedby the difficulty in obtaining sufficient quantities of thisprotein because of its low abundance in cells. Attemptsto express the RB protein by introducing the codingsequence of the RB gene into a bacterial expressionvector have been only partially successful. Bacteriallyproduced proteins have poor solubility, and the majorproduct is an Mr 56,000 protein derived from internalinitiation of translation.4 Another drawback of using thebacterial expression system is that bacterial cells areunable to modify eukaryotic proteins, and analysis ofsuch proteins could be misleading if posttranslationalmodifications are required for the normal function of theprotein. This problem could best be circumvented bythe alternative approach of expressing the cloned genein a eukaryotic system.

The baculovirus Autographa ca!ifornica nuclear poly-hedrosis virus has been shown to be suitable as a helper-independent viral expression vector for the high-levelproduction of recombinant proteins in cultured insectcells (23). This virus propagates in cultured Fall Armyworm Spondoptera frugiperda cells and has a strong tern-porally regulated promoter ofthe polyhedrmn gene whoseproduct represents 50% or more of total cellular proteinsduring a lytic infection. By in vivo recornbination, the

Received 5/30/90.I This work is supported by grants from the National Cancer Institute

(CA 49649) and the Council for Tobacco Research (CTR249 to E. V-H. P.

1., and by a National Eye Institute grant (EY05758) to W-H. L. N. P. W. is

a trainee supported by a National Eye Institute training grant (EY57980)to W-H. L.

2 To whom requests for reprints should be addressed, at Departmentof Pathology-0612, University of California at San Diego, La Jolla, CA

92093.

3 The abbreviations used are: cDNA, complementary DNA; kb, kilo-

base; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel ebectro-

phoresis; PAP, potato acid phosphatase; MOl, multiplicity of infection;PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline;DTT, dithiothreitol; AcNPV, Autographa californica nuclear polyhedrosis

virus; Sf9, Spodoptera frugiperda cells.

4 S. Huang et a!., unpublished observation.

Bamfil

aaaaaaacctataaata CGGATCC CGCCGCGGA A AGGCGTC ATG

Polyhedrin BamHl RB sequences L_._e...promoter linker

Fig. 1. Construction of bacubovirus expression vector for ppl 10*B syn-

thesis. pRB44-2 consists of the complete RB cDNA coding sequencefrom nucleotide 116 to 2935 subcloned into the BamHI site of pGEM1.

pAcYM1 contains the -7-kb EcoRl fragment of the viral DNA sequenceflankingthe polyhedrin gene in which the leader sequence remains intact,but all of the polyhedrin coding sequences except the first A of the ATG

are replaced by a BamHl linker. The recombinant bacubovirus vector,pAcYM1/RB2.8, containing pobyhedrin promoter-RB cDNA fusion, was

constructed by inserting the RB 2.8-kb BamHI fragment into the BamHlsite of pAcYM1 so that the transcription of the RB gene will be under thedirect control of the polyhedrin promoter. The sequence at the junctionof the fusion is shown at bottom; lower case letters, polyhedrin promoter;

upper case letters, RB cDNA sequence; underlining, the BamHl linker.

Arrow, the translation of the fusion gene utilizing the ATG of the RB(nucleotide 139); a�’, the first A of the translation start codon ATG of

polyhedrin gene.

430 Baculovirus Production of RB Protein

coding sequence of a foreign gene can easily be placedunder the transcriptional control of the polyhedrmn pro-moter, resulting in a high level of expression. In addition,such proteins may be correctly folded and contain ap-propriate posttranslational modifications like those pro-teins in the original higher eukaryotes (24). To test thefeasibility of expressing functional RB protein by thebaculovirus system, cloned human RB cDNA, containingthe complete coding sequence of the RB gene, wasintroduced into the AcNPV expression vector, and therecombinant viruses were propagated in insect cells.Here we report the successful expression of humanppi i0� at high level by this host-vector system in whichthe protein produced is phosphorylated and correctlytargeted to the nuclei of infected cells. We also describethe method for the purification of RB protein and findthat this purified protein can bind DNA and form aspecific complex with SV4O T antigen in the same wayas the authentic human ppl iORB. The prompt nucleartranslocation of the protein after microinjection furthersuggests the active nature of the purified RB protein.

Results

Construction of Recombinant Baculovirus Containing theRB Gene. In an attempt to achieve maximal productionof the RB protein in the baculovirus expression system,the recombinant transfer vectors were constructed withdeletion of most of the 5’ noncoding sequence of theRB gene. By site-specific mutagenesis, two BamHl siteswere introduced into the RB cDNA at nucleotides 116

Transcription and 2935 (1 3) to facilitate the following construction of

the recombinant transfer vector (see HMaterials andMethods”). As shown in Fig. 1, the resulting pAcYM1/RB2.8 encodes mRNA that contains the entire (60 basepairs) polyhedrin 5’ noncoding sequence fused to 23base pairs of the 5’ untranslated region of the RB cDNAfollowed by the complete coding sequence. This recom-binant gene contains no ATC codons upstream of theauthentic RB initiation site at nucleotide 139 and thusencodes a nonfusion full-length RB protein.

Expression of Exogenous RB Protein in AcNPV-Y4 RB-infected Insect Cells. To determine whether the AcNPVpolyhedrmn promoter could drive the expression of hu-man RB gene in heterologous invertebrate cells, Sf9 cellswere infected with plaque-purified AcNPV-Y4 RB. Fortyh after infection, lysates of the infected cells were col-lected and immunoprecipitated with anti-RBO.47 anti-body. Samples were then subjected to SDS-PACE, fol-owed by Western blot analysis. As shown in Fig. 2A,

immunoblotting with pMC3-245 rnonoclonal antibodyrevealed the appearance of full-length RB protein similarto that of the mammalian cells (Lane 1 ) in extracts of cellsinfected with AcNPV-Y4 RB (Lane 3), but not in those ofmock or wild-type AcNPV-infected cells (Lanes 2 and 4).

The production of the RB protein was monitored dur-ing the postinfection period to determine the optimaltiming for harvesting the cells. As shown in Fig. 2B, RBprotein production could be detected at 24 h after infec-tion and significantly increased during the following i2h. The level of protein production was maintainedthrough ‘-�‘72 h of infection, at which time significant virallysis of cells began. To minimize protein degradationassociated with cell lysis, infected cells were routinelyharvested around 40 h postmnfection.

Nuclear Localization and Posttranslational Phosphor-ylation of the Exogenous RB Protein in Insect Cells. TheRB gene encodes a nuclear phosphoprotein of Mr

ilo,000 (13). To determine whether RB protein pro-duced in insect cells with the baculovirus was targetedto the nucleus, AcNPV-Y4 RB-infected Sf9 cells wereimmunostained with anti-RBO.47 antibody 40 h afterinfection. As shown in Fig. 3, infected cells containedunusually large nuclei characteristic of the cytopathiceffect of baculovirus infection. When mock-infected orwild-type AcNPV-infected Sf9 cells were incubated withanti-RBO.47 antibody, no staining was observed (Fig. 3Aand data not shown). However, intense staining wasfound exclusively in the nuclei of cells infected withAcNPV-Y4 RB (Fig. 3B). Analysis by SDS-PACE and West-em blotting of the nuclear and cytoplasmic extracts fromAcNPV-Y4 RB-infected Sf9 cells confirmed that the ex-ogenous RB protein is present predominantly in thenuclear fraction (data not shown).

Phosphorylation of RB protein occurs at multiple serineand threonine residues and accounts for the molecularweight heterogeneity of RB protein in the SDS-PACE (16,25). To determine whether RB protein produced in the

RBpp110

� �p110 a-

RB

pp110

RB � -

pl1O-� -..�. -

A.. .‘�

5,

- -...

p

\

Ba, I , I

Fig. .1. Intracellular localization of RB protein expressed in inse t ( oIls

by immunostaining. A, mock-infected Sf9 cells; B, A NPV-Y4 RB-inle tedSf9 cells.

Cell Growth & Differentiation 431

A BHours post-infection

1 2 3 4 24 36 48 60 72

Fig. 2. Identification of ppl lO�� in AcNPV-Y4 RB-infected insect cells by Western blot analysis. A, cellular extracts were prepared 40 h p�stinfection

from mock-infected (Lane 2), AcNPV-Y4 RB-infected (Lane 3), or wild-type AcNPV-infected Sf9 cells (Lane 4). Mobt-4, a human leukemia cell line, was

used as the control (Lane 1). B, cellular extracts from AcNPV-Y4 RB-infected cells were prepared at different time points postinfeoion in order to

determine the optimal timing for RB protein production. The lysates were immunoprecipitated with anti-RBO.47 antibody and immunoblotted withpMG3-245 monoclonal antibody. p1 10R8 and ppl 10R8, unphosphorylated and phosphorybated RB proteins, respectively.

insect cells undergoes phosphorylation posttransla-tionally, AcNPV-Y4 RB-infected Sf9 cells were metaboli-cally labeled with [35S}methionine or 32P, for 3 h at 40 hafter infection. Cell extracts were subjected to immuno-precipitation and analyzed by SDS-PAGE, followed byautoradiography (Fig. 4, Lanes 2 and 4, respectively). Inparallel, immunoprecipitable RB protein from the sameextracts was treated with PAP to test the effect of de-phosphorylation on RB protein mobility in SDS-PACE.After dephosphorylation, the 35S-Iabeled RB protein wasreduced from a doublet to a single band of Mr 1 10,000

(Fig. 4, Lane 2 ‘), and radioactivity was almost completelyreleased from 32P-labeled RB protein (Fig. 4, Lane 4’).Dephosphorylation analysis by Western blotting of ly-sates from unlabeled cells infected with AcNPV-Y4 RBalso showed the same band reduction pattern after PAPtreatment (Fig. 4, Lane 6 and 6’). These observationsindicated that RB protein produced in insect cells wasphorphorylated, and the modification also accounted forthe molecular weight heterogeneity of this RB proteinobserved in the SDS-PAGE (Figs. 2 and 4).

Purification of RB Protein from Infected Insect Cells.Sf9 cells were infected with AcNPV-Y4 RB at an MOI of1 .0, and 40 h after infection, cellular lysates were pre-pared. Under this condition, the total level of RB proteinexpressed in the baculovirus system was approximately17-18 mg/liter of infected insect cell culture (‘-‘10” cells).As shown in Table 1, 90% (16 mg) of the RB proteinexpressed was found in the supernatant after cell disrup-tion, whereas 10% remained in the insoluble fraction.The RB protein could readily be detected in the cellularlysate (Fig. 5, Lane 2), as it represented 2.3% of the totalcellular proteins. Following the one-step immunoaffinitychromatographic purification, approximately 1 3.5 mg ofproteins could be recovered from the alkaline eluates ofthe column. To estimate the purity of the eluted RBprotein, an aliquot of the eluates corresponding to 2.5 xiO� cells was analyzed by SDS-PAGE and Coomassiebrilliant blue staining. As judged by densitometry, thissingle purification step proved to be efficient, resulting

4�o

bi � �

,

,� __ ___ir i i� ir I � �

1’ 2 2’ 3 3’ 4 4’ 5 5’ 6 6’

RBPP!!0 -1

�P110

II

Table 1 Puritication (it re ombinant RB protein from l)aculovirus-in-

tected insv I cellskd

1 80 -

116-

84 -

58 -

48 -

36-

26 -

a Protein (luanlitation I)y the method ot Bradford )Bio-Rad(.b Protein quantitation I)y Western blot and densitometry.�:Protein quantitation by Micro-BCA (Pierce( and spectrophotomotry.

‘I Protein quantilation by Coomassie brilliant blue staining and densitom-

etry.

a-

Fig. 5. Immunoaffinity chromatographic purification of ppl 10’#{176}.Crudelysates from 1 x iO�, mock- (Lane 1) or AcNPV-Y4 RB-infected (Lane 2)Sf9 cells as well as an abiquol (corresponding to 2.5 X 10� infected cells)

of the eluates from the pMG3-245 anti-RB immunoaffinity chromatogra-phy (Lane 3) were analyzed by electrophoresis on a 10% SDS-polyacryl-amide gel, followed by Coomassie brilliant blue staining. Arrow, theposition of the RB protein with expected molecular weight.

432 Bacubovirus Production of RB Protein

St01)Total

pri)I(’inmg)

RB

I)rotein

(mgi

Yield

)%)

Purifi ation

fold

Purity(%)

Cellular extra( I 67tY 1 6b 90i� 1 .0 2.3

PMG3245 immu- 13.5’ 12.8” 72” 41.3 5

noattinitv (

umn

in a preparation of RB protein with 95% purity (Fig. 5,Lane 3), a 72% yield, and a 41.3-fold of purification(Table 1).

DNA-binding Activity and Specific Complex Forma-tion with SV4O I Antigen. To date, two biochemicalproperties of the RB protein have been described. Oneis its ability to bind DNA intrinsically (13, 26), and theother is its ability to form specific complexes with onco-proteins of several DNA tumor viruses (27-29). The RBprotein purified from baculovirus-infected insect cellswas tested for these two known biochemical properties,which have been implicated in its biological functions.As shown in Fig. 6, DNA binding was assayed by South-western analysis, in which identical amounts of the trpE-RB fusion proteins as well as the purified RB protein frominsect cells were separated by 10% SDS-PAGE. Thequantity of loaded protein was confirmed by Coomassiebrilliant blue staining (Fig. 6A). Another gel run in parallelwas electrotransferred to nitrocellulose membrane fol-lowed by incubation with 32P-labeled DNA. DNA boundto the protein was then analyzed by autoradiography(Fig. 68). As previously shown, fusion protein RB19-27,which contains the major domain for interacting withDNA (26), has a 20-fold higher affinity for DNA thaneither of two subregions, RB19-22 and RB23-27 (Fig.6B; compare Lane 3 with Lanes 1 and 2), whereas the

4�, Fig. 4. Phosphorylation of RB

protein produced in insect cellsand dephosphorylation analysis.Forty h after infection withAcNPV-Y4 RB, Sf9 cells weremetabolically labeled with [“SI

methionine or ‘2P, for 3 h. Molt-4 was included as the control,

and cellular lysates were thenimmunoprecipitated with anti-RBO.47 antibody. The 355� and

‘2P-babeled RB protein immunecomplexes were separated by

SDS-PAGE before (Lanes 1, 2, 3,and 4) or after treatment with

potato acid phosphatase (Lanes1’, 2’, 3’, and 4’) and analyzed

by autoradiography. Similar de-

phosphorylation experimentsusing lysates from unlabeledcells were performed and sub-jected to Western blot analysisbefore and after treatment with

potato acid phosphatase (Lanes5, 6, and 5 ‘, 6 ‘ , respectively).

1 2 3

purified full-length RB protein exhibited a strong DNA-binding activity similar to that of RB19-27 (Fig. 68, Lane

4). DNA-binding activity of the purified RB protein frominsect cells was also demonstrated by retention of theprotein by DNA-cellulose and its subsequent elutionfrom the column at approximately 400 mr�i NaCI (datanot shown).

To test the ability of the purified RB protein to form aspecific complex with SV4O T antigen, equal amounts ofRB protein and T antigen were mixed, and aliquots ofthe mixture were immunoprecipitated with either anti-RBO.47 antibody or anti-T antibody PAB419. As shown

B

kd

116-

84 -

58-

1 234

�- .�

A

kd

116-

84 -

58-

I antigen -� JTi�_ �- _ 1-F- -s--�

1 234

‘pp

..#{248}__1-� � � o:..� p110

Cell Growth & Differentiation 433

Fig. 6. Southwestern DNA-binding assay. Six �g of purifiedtrpE-RB fusion proteins as wellas the purified bacubovirus-ex-

pressed ppl 10R8 were ana-

lyzed by 10% SDS-PAGE. A,Coomassie brilliant blue stain-ing. B, a parallel gel was elec-

trotransferred onto the nitro-cellulose paper, and the blot

was incubated with “P-labeled

DNA fragments and analyzedby autoradiography. Lane 1,RB19-22; Lane 2, RB23-27;

Lane 3, RB19-27; Lane 4, pun-fied RB protein from AcNPV-Y4 RB-infected insect cells.

Fig. 7. Complex tormation of

baculovirus-expressed RB pro-tein with SV4O T antigen. Pun-

fied baculovirus-expressed RBprotein was mixed with pun-fied T antigen in vitro. Identical

aliquots of the mixtures werethen immunopnecipitated withPAB149(Lane 2) on anti-RBO.47

(Lane 3) and analyzed by West-

em blotting. Lanes 1 and 4, pu-nified SV4O T antigen immuno-

pnecipitated with PAB419, and

punified bacubovirus-expnessedRB protein immunoprecipi-

tated with anti-RBO.47 anti-body, respectively.

1 2 3 4

in Fig. 7, mixing of RB protein with T antigen in vitroresulted in the coimmunoprecipitation of RB protein withPAB419 (Lane 2), as well as the coimmunoprecipitation

of I with anti-RBO.47 antibody (Lane 3). These datademonstrated that RB protein from baculovirus-infectedinsect cells can form a specific complex with SV4O Tantigen.

Nuclear Translocation of the Purified RB Protein inMammalian Cells. After showing that the purified proteinretained the two known biochemical activities of RB invitro, we were also interested to see how this purifiedprotein would behave in vivo. Purified RB protein wasinjected into the cytoplasm of Saos-2 cells, an osteosar-coma cell line which contains a defective RB gene withdeletion of exons 21-27 and encodes a C-terminal trun-cated RB protein (p9S) (30). This protein, located in thecytoplasm in minute amounts, is not recognized by theanti-RBO.47 antibody used here, as the antibody is di-rected against the C-terminus of RB protein (31). Imme-diately after injection, cells were fixed and subjected toimmunostaining analysis. As shown in Fig. 8, intensestaining of the nucleus of the injected cell was found(arrow) as compared to that of the uninjected control,indicating the rapid transport of the injected protein intonuclei. Since RB protein has been known as a nuclear

protein, this prompt and correct nuclear translocation ofpurified protein after microinjection further suggestedthat the protein may be active in vivo.

Discussion

In this study, we demonstrate that the human retinoblas-toma gene product can be expressed efficiently underthe transcriptional control of the baculoviral polyhedrinpromoter. The attempt to express RB protein at highlevels has long been regarded as difficult, since it wassuspected that RB protein might hinder or even be“toxic” to the growth of cells. The transcription of foreign

genes from the polyhedrin promoter occurs late in infec-tion, following production of extracellular viral particlesand the shut-off of cellular and most viral genes; thebaculovirus-insect cell system is therefore advantageousfor the synthesis of proteins like RB which may be detri-mental to cell growth when overproduced. Another gen-eral advantage of this system is the similarity in proteinprocessing pathways of insect and mammalian cells. TheRB protein produced is shown to be correctly targetedto the nuclei of insect cells, implying that the mammaliannuclear translocation signals are also recognized by insectcells. Although glycosylation of recombinant proteins in

.4

“�

a

434 Baculovirus Production of RB Protein

5 N. P. Wang et al., unpublished observation.

fig. 8. Nuclear translocation ot the purified RB protein alter mi-

( noinle( ti(in into cytoplasm of Saos-2 cells. Saos-2 cells were in�e tedwith Purified RB protein and sul)jected to immunostaining analysis.Arrno.’, the intense staining of the nucleus after micnoinle( tion as

( onip�irr’d to that ol uninjected ( oIls.

the baculovirus expression system seems limited to the0-linked and N-linked oligosaccharides of the high man-nose type (24, 32-34), appropriate phosphorylation offoreign proteins has been reported for the expression ofc-myc (35), bcr-abl (36), and HTLV-b p40k (37). RB proteinhas previously been shown to be phosphorylated but notglycosylated (1 3), making the baculovirus expression sys-tem quite suitable for the production of functional RBprotein. Here we show that the RB protein produced ininfected insect cells is posttranslationally phosphorylatedand that multiple bands can be differentiated on theWestern blotting analysis, just as the authentic mamma-han RB protein. However, as judged by the band inten-sity, un- and hypophosphorylated forms are predominantwhen compared to the hyperphosphorylated RB protein.We do not know at this point whether this phenomenonis a reflection of the cell cycle status of the populationduring a viral lytic infection or is simply due to theinsufficient phosphorylation of the protein by insect ki-nases because of the massive amount of exogenous RBpresent in cells. Precise mapping of phosphorylation sitesin the RB protein will also be necessary in order todetermine whether the phosphorylation patterns aretruly identical to that of mammalian protein.

The total level of recombinant RB protein expressedin the baculovirus system is about 17-18 mg/liter ofinfected insect cell culture (‘�-10� cells). This level ofexpression is comparable to other mammalian proteinsproduced by this system, such as 10-20 mg/liter forinterleukin 2 (38) and 4-5 mg/liter for P210 BCR-ABL(36). The high level of expression of RB protein is prob-ably contributed by using a recombinant transfer vectorcontaining the intact polyhedrin 5’ untranslated regionfused with the RB cDNA deprived of most of its 5’noncoding region. This sequence of the RB mRNA ishighly G+C rich, which may favor the formation of stablesecondary structures (10). These structures, when pres-ent in front of an initiation codon, are thought to decreasethe translational efficiency of the corresponding mRNA

(39, 40). Five- to 10-fold enhancement of the in vitrotranslation of RB mRNA has been demonstrated by thereplacement of RB 5’ untranslated sequence with that ofthe alfalfa mosaic virus RNA4 or �-globin mRNA, furthersuggesting the potential adverse effect of RB 5’ noncod-ing sequence on the translation (31). The presence of

long 5’ untranslated sequences of the foreign genes hasalso been shown to affect the recombinant proteinexpression in the baculovirus system (24, 36). Since thepolyhedrin promoter is very A+T rich, it is suggested thatthe long and G+C-rich 5’ noncoding sequence betrimmed from the RB cDNA prior to the insertion intothe transfer vector for the optimal expression of ppl 10’��.

Several different protocols for the elution of RB proteinfrom the affinity column have been tested in an attemptto minimize the denaturation of protein during the pun-fication process. Since not much is known of the biolog-cal functions and biochemical properties of RB protein,

the only two parameters that can be used as measuresof the integrity of purified protein are the activities ofDNA binding and complex formation with SV4O T anti-gen. It was found that the present elution condition using20 m�i tniethylamine at pH 10.8 seemed optimal inpreserving both biochemical properties of the protein.Rapid nuclear translocation of the purified protein fromthe cytoplasm after microinjection further suggested thatthe protein was active under this elution condition. Flu-tion of the protein at extreme pH (200 mtvt glycine, pH2.3, or 100 mM tniethylamine, pH 1 1 .5) tended to dena-tune the protein in that the aforementioned two activitieswere greatly diminished and were also evident by theformation of insoluble aggregates after long-term storage.

Despite a previous report (25) that only the unphos-phorylated RB protein can bind SV4O T antigen in D2C2cells, a stable transformant of monkey kidney cell lineCV1-P by SV4O T antigen, we found that certain hypo-phosphorylated forms of the RB protein were also ableto form complexes with the SV4O T antigen. This hasbeen reproducibly demonstrated with the in vitro mixingof I antigen with purified RB protein from AcNPV-Y4RB-infected insect cells or with Molt-4 lysates. The samephenomenon has also been observed when using Coscells for in vivo complex formation.5 Since phosphoryla-tion of the RB protein oscillated during the cell cycle ina phase-specific manner (41-43), and the complex for-mation between RB and viral oncoproteins has beenimplicated in the transforming activity of these DNAtumor viruses (27-29), the significance of this associationbetween hypophosphorylated RB protein and 5V40 Tantigen awaits future elucidation.

The availability of a large amount of soluble, intact,and presumably active RB protein by the baculovirus-insect cell system represents a major advance for futurestudies of the biochemical and biophysical properties ofthe RB gene product. Possible applications include analy-sis of associated cellular proteins, isolation of the specificDNA sequence with which it interacts, and the three-dimensional structural studies of the RB protein by X-raycrystallography. The elucidation of the biological func-tion of RB in cancer suppression can also be facilitated:its possible involvement in cell growth and differentia-

Cell Growth & Differentiation 435

tion, directly tested by microinjection, is now underactive investigation.

Materials and MethodsCells. Sf9, a clonal isolate of Spodoptera frugiperda IPLB-Sf21-AE (44), was grown as monolayer or suspensioncultures at 27#{176}Cin Grace’s insect medium supplementedwith 3.33 g/liter of yeastolate, lactalbumin hydrolysate(GIBCO), and 10% heat-inactivated fetal bovine serum(GIMINI) (23). In large-scale preparation of cellular ly-sates, spinner cultures of Sf9 cells were grown in EX-CELL 400 serum-free defined medium (J. R. Scientific).Molt-4 cells, a human T-cell leukemia line (45), werecultured in suspension in RPMI 1640 supplemented with20% calf serum. Saos-2 cells, an osteosarcoma cell line,

were grown in Dulbecco’s modified Eagle’s mediumsupplemented with 7.5% fetal bovine serum.

Construction of Recombinant Plasmids and Recombi-nant Baculoviruses. The transfer vector pAcYM1, whichhas all the upstream sequences of the polyhedrin geneincluding the A of the initiating ATG codon followed bya unique BamHl site, has been described by Matsuura eta!. (46). pRB44-2 contains the complete RB cDNA codingsequence from nucleotides 1 16 to 2935 subcloned intothe BamHI site of plasmid pGEM1 (Promega). The recom-binant baculovinus vector, pAcYM1/RB2.8, was con-structed by inserting the 2.8-kb BamHI fragment frompRB44-2 into the BamHl site of pAcYM1 in the correctorientation so that the transcription of the RB gene isunder the direct control of the polyhedrin promoter (Fig.1). Transfer of RB cDNA from the recombinant plasmidto the viral genome was achieved by cotransfectingpAcYM1/RB2.8 DNA with wild-type Autographa ca!ifor-nica nuclear polyhedrosis virus DNA by lipofection (Be-thesda Research Laboratories). The recombinant viruses,in which the polyhedrin gene had been inactivated byallelic replacement with the RB gene through homolo-gous recombination, were identified by the distinctplaque morphology, as they showed no polyhedrin oc-clusion bodies in infected cells (23). The viruses weresubjected to three rounds of plaque purification to obtaina pure stock of RB-containing baculovirus designatedAcNPV-Y4 RB.

Detection of Expression of the RB Protein. AcN PV-Y4RB was used to infect Sf9 cells at an MOI of 0.5. At 24,36, 48, 60, and 72 h postinfection, 5 x iO� cells werelysed at 4#{176}Cin i ml of lysis buffer (50 mr�i Tnis-HCI, pH7.4-0.2% Nonidet P-40-1 mM EDTA-100 misi NaCl-50 m�iNaF-i mM PMSF), and the lysates were clarified by cen-trifugation (4#{176}C,20,000 x g) for 5 mm. Lysates were thenincubated with anti-RBO.47 antibody (31), and immuno-precipitates were separated by 7.5% SDS-PAGE. Proteinswere then transferred to nitrocellulose paper as de-scribed (47). After overnight blocking, the nitrocellulosepaper was incubated with pMG3-245 anti-fRB monoclo-nal antibody for 3 h, followed by alkaline phosphatase-conjugated goat anti-mouse lgG and colonigenic sub-strates as described (27).

Immunostaining Analysis. After 40 h of either mock,wild-type AcNPV, or AcNPV-Y4 RB infection, Sf9 cellswere seeded on poly-L-lysine (Sigma)-coated chamberslides (Miles Scientific) and incubated overnight. Slideswere washed with PBS between each of the followingsteps: cells were first fixed with 4% formaldehyde in 0.04M phosphate buffer (pH 7.4) for 20 mm or with acetone

(-20#{176}C)for iO mm and immersed in i% H2O2 in meth-anol for 10 mm. Fixed cells were preincubated with 2%normal goat serum in PBS for iO mm and then incubatedovernight with rabbit anti-RBO.47 antibody diluted in0.02% Triton X-iOO. After washing, biotinylated goat anti-rabbit lgG (Tago, Burlingame, CA) was added. One hlater, cells were incubated with AB complex conjugatedwith horseradish peroxidase (Vector Laboratories, Burlin-game, CA) for 45 mm and then incubated with substrate(0.05% 3,3 ‘-diaminobenzidine tetrahydrochlonide and0.01% H2O2 in 0.05 M Tnis-HCI, pH 7.6) (Sigma). Reac-tions were stopped 3-5 mm later by washing cells withPBS. Cells were photographed with a Nikon diaphoto-microscope.

Radiolabeling of Sf9 Insect Cells and Dephosphoryla-tion Analysis. At 40 h postinfection, Sf9 cells (3 x 106) in60-mm dishes were incubated with Dulbecco’s modifiedEagle’s medium lacking either methionine or phosphateand supplemented with 10% fetal calf serum for 30 mm.The cells were then metabolically radiolabeled by sup-plementing with 0.25 mCi/mI [35S]methionine (1 i 34 Ci/mmol; NEN) or with 0.25 mCi/mI 32P (carrier-free; ICN)for 3 h. Cell extracts were then prepared in lysis buffer(50 m� Tris-HCI, pH 7.4-0.2% Nonidet P-40-i m�i EDTA-100 mM NaCl-50 mM NaF-i mt.i PMSF), and immunopre-cipitation with anti-RBO.47 antibody was performed aspreviously described (31).

Two-thirds of the immunoprecipitated RB protein from355� or 32P-labeled as well as unlabeled cell lysates wassubjected to potato acid phosphatase (Boehninger) de-phosphorylation analysis (16). Immune complexes con-taming the RB protein were incubated with i .5 units ofPAP in reaction buffer [20 mM (2-[N-morpholino]ethane-sulfonic acid) sodium salt, pH 5.5-100 m� NaCl-i mt’.iMgCl2-50 zM leupeptin] for 60 mm at 37#{176}C.After thereaction, RB protein was analyzed by 7.5% SDS-PAGEfollowed by autoradiography or Western blotting accord-ingly.

Preparation of the lmmunoaffinity Column. Proce-dures for the construction of immunoaffinity columnfollowed the methods described by Schneider et a!. (48)and Simanis and Lane (49) with minor modification. Twoml of protein G-agarose (Genex) were packed in a Bio-Rad column and washed with 0.Oi N HCI followed bythe binding buffer (0.1 M sodium acetate, pH 5.0-0.1 M

NaCI). Fifteen mg of anti-fRB monoclonal antibody(pMG3-245) were applied to the column twice to allowbinding. The column was then washed extensively with0.i M borate buffer, pH 9.0, and the beads were nesus-pended in 20 ml of the same buffer. Dimethylpimelimi-date dihydrochloride (Sigma) was added to a final con-centration of 40 mM, and the mixture was agitated for ih at room temperature for the cross-linking reaction totake place. After washing, the remaining reactive groupsof the beads were blocked with 40 m�i ethanolamine-HCI in 20 ml of 0.1 M borate buffer, pH 8.0, for 10 mmat room temperature. The column was then washed with0.2 M glycine, pH 2.3, and neutralized with Tris buffer(50 mt�i Tnis-HCI, pH 7.4-100 m�i NaCl-1 m�i PMSF-i mt�iEDTA), in which it was stored until required. By meas-uning A280 of the original monoclonal antibody sampleand that of the flow-through fractions in subsequentsteps, it was estimated that approximately iO mg ofpMG3-245 were coupled to the 2 ml of protein G-agarose beads.

Purification of pp1 lOla from Infected Insect Cells. Sf9

436 Baculovirus Production of RB Protein

cells were infected with AcNPV-Y4 RB at an MOI of 1.0and cultured in suspension (i x 106 cells/mI; 1000 ml).After 40 h of infection, the cells were pelleted by low-speed centnifugation, washed, and resuspended in anextraction buffer containing 50 mt�i Tris-HCI, pH 7.4-0.2% Nonidet P-40-1 mr’�i EDTA-100 m�i NaCl-iO% (v/v)glycerol-i m� DTT-1 mt�’t PMSF-25 zg/ml leupeptin-50units/mI aprotinin. After a 1 5-mm incubation on ice, thesample was clarified by centnifugation (10,000 x g, 4#{176}Cfor 10 mm), and the RB-containing supernatant was col-lected. Immunoaffinity chromatography of ppliORB wascarried out on a 2-mI-volume column containing anti-fRB monoclonal antibody (pMG3-245) linked to proteinG-agarose as described above. After passing the super-natant through the column four times, the column waswashed sequentially with 200 bed-volumes of each ofthe following: lysis buffer, lysis buffer containing 500 mr�iNaCI, and washing solution (200 mt�i NaCI-1 mi’�i EDTA-i mM DTT-1 mM PMSF-10% glycerol). Bound proteinswere then eluted from the column by alkaline elutionbuffer containing 20 mt�i tniethylamine, pH 10.8-200 mr�iNaCl-i mM EDTA-i mivi DTT-i m�i PMSF-10% glycerol.One-mi fractions were collected, immediately neutral-ized with one-twentieth volume of 1 M Tris-HCI (pH 7.5),and stored at -70#{176}Cin iO% glycerol.

Protein Quantitation. The amount of total protein inthe elution fraction of the immunoaffmnity column wasdetermined by Micro-BCA assay (Pierce). The elutedprotein sample was then analyzed by SDS-PAGE, and theamount of RB protein in the eluates was estimated byCoomassie brilliant blue staining followed by densitom-etry. The amount of total protein in the cellular extractwas measured by the method of Bradford (Bio-Rad) (50).To quantitate RB protein in cellular lysates, Westernblotting was performed using serially diluted purified RBprotein as standard followed by densitometric compari-son of the band intensity (Table 1).

Southwestern DNA-binding Assays. Protein blottingwas performed as described (47). Incubation of blots withradiolabeled DNA followed the protocols described byBowen et a!. (51). The whole procedure was carried outat room temperature. Blots were rinsed briefly with waterand then washed three times with 6 M urea-0.2% NonidetP-40 (20 mm each), followed by four washes (30 mmeach) with DNA-binding buffer (iO mt�i Tnis-HCI, pH 7.0-1 mM EDTA-50 mM NaCI-0.02% bovine serum albumin-0.02% Ficoll 400-0.02% polyvinyl pyrrolidone). The blotswere then incubated for 30 mm in DNA-binding buffercontaining 32P-labeled DNA. pGEM1 DNA linearized byEcoRl was labeled with a-32P-deoxynucleotides (Amer-sham, >3000 Ci/mmol) by random priming and was usedas probe. After hybridization, blots were washed threetimes (10 mm each) with DNA-binding buffer, air dried,and analyzed by autoradiography. trpE-RB fusion pro-teins were included as controls and have been describedin detail by Wang et a!. (26). Each trpE-RB fusion proteinwas named according to the exons of the RB gene thatthe protein contains, i.e., RB19-22, RB23-27, and RB19-27 spanned the regions of ppllORB from exon 19 to 22(amino acid 612-775), exon 23 to 27 (amino acid 776-928) and exon 19 to 27 (amino acid 612-928), respec-tively.

SV4O T Antigen Binding Assay. SV4O I antigen waspurified by immunoaffinity chromatography from Ad-SVXi-infected 293 cells (52, 53), and the anti-T monoclonalPAB4i9 antibody was from Oncogene, Inc. The complex

formation assay was performed as previously described(27) with minor modifications. Briefly, 800 ng of baculo-virus-expressed RB protein were mixed with 1 ml of EBCbuffer (50 mM Tnis-HCI, pH 8.0-120 mt’�i NaCI-0.5% Non-idet P-40) containing 1 mt�i PMSF-25 zg/ml leupeptin-50units/mi aprotinin. Eight hundred ng of purified T wereadded to the mix, and the mixture was incubated on icefor 90 mm. Aliquots of the mixture were immunoprecip-itated with either anti-RBO.47 or PAB419 antibody andsubjected to Western blotting analysis. Blots were se-quentially reacted with pMG3-245 followed by PAB419.After incubating with alkaline phosphatase-conjugatedgoat anti-mouse lgG, the blots were then developed withcolorigenic substrates.

Microinjection. For microinjection, purified RB proteinwas dialyzed into injection buffer containing 20 mts’t Iris-HCI, pH 7.4-10 mM KCI-0.1 mt.i EDIA-0.1 mt�t DTI-2%glycerol to a final concentration of 0.5 mg/mI. Saos-2cells growing on glass chamber slides were microinjectedessentially as described previously (54), using glass cap-illary needles (Eppendonf). An Eppendorf micromanipu-lator equipped with a vacuum and pressure device andan inverted phase-contrast microscope (Nikon) wereused for micromanipulation of the capillary and visuali-zation of the microinjection process. After microinjec-tion, cells were immediately fixed by 4% formaldehydein 0.04 M phosphate buffer (pH 7.4) and subjected toimmunostaining analysis.

Acknowledgments

We are very grateful to Dr. Ann-Ping Tsou for her valuable technical

advice and Dr. Tony Hunter for critical comments on the manuscript.We also thank Drs. David Goodrich, Joachim Schnier, Peter Scully, andRobert Bookstein for their suggestions.

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