high and compact formation of baculoviral polyhedrin-induced inclusion body by co-expression of...

ARTICLE

High and Compact Formation of BaculoviralPolyhedrin-Induced Inclusion Bodyby Co-Expression of BaculoviralFP25 in Escherichia coli

Lin Li,1,2 Young Soo Kim,1,3 Dong Soo Hwang,1,3 Jeong Hyun Seo,1,3 Hee Jung Jung,1,3

Juan Du,2 Hyung Joon Cha1,3

1Division of Molecular and Life Sciences, Pohang University of Science and Technology,

Pohang 790-784, Korea; telephone: þ82-54-279-2280; fax: þ82-54-279-2699;

e-mail: [email protected] Key Laboratory of Agricultural Microbiology, School of Life Science and Technology,

Huazhong Agricultural University, Wuhan, China3Department of Chemical Engineering, Pohang University of Science and Technology,

Pohang, Korea

Received 21 June 2006; accepted 24 August 2006

Published online 26 September 2006 in Wiley InterScience (www.interscience.wiley.c
om). DOI 10.1002/bit.21193
ABSTRACT: Previously, we found that baculoviral polyhe-drin (Polh) can successfully be used in Escherichia coli as afusion partner for the expression of special foreignproteins as inclusion bodies, and the resulting, easilyisolatable Polh-induced fusion inclusion bodies had almostthe same characteristics as the native Polh. Here, we inves-tigated the effects of co-expression of baculoviral FP25protein on Polh-induced inclusion-body production in anE. coli expression system, as FP25 is known to be involvedspecifically in polyhedra formation. Using several analyticaltools, including SDS–PAGE, pronase proteolysis, solubiliza-tion under alkaline conditions, and electron microscopy, wefound that co-expressed FP25 was associated with Polh-induced inclusion bodies and that its co-expression led toformation of compact inclusion bodies as well as highproduction levels. We confirmed that FP25 co-expressioninduced higher production levels of other heterologousprotein, antimicrobial peptide Hal18, fused with aggrega-tion-prone Polh. Therefore, co-expression of baculoviralFP25 can be promisingly used to increase the levels ofbaculoviral Polh-fused foreign proteins, especiallyharmful proteins, expressed as inclusion bodies in an E. coliexpression system.

Biotechnol. Bioeng. 2007;96: 1183–1190.

� 2006 Wiley Periodicals, Inc.

KEYWORDS: baculoviral FP25; baculoviral polyhedrin;inclusion body; fusion protein; Escherichia coli

Correspondence to: H.J. Cha

� 2006 Wiley Periodicals, Inc.

Introduction

Nucleopolyhedrovirus (NPV) is a genus of Baculoviride,a large family of enveloped, double-stranded DNA-containing viruses that are pathogenic to arthropods,particularly members of the Lepidoptera (Federici, 1997).The rod-shaped virions of NPVs are occluded into a largeand dense paracrystalline matrix known as a polyhedra, ofwhich the major protein component is the proteinaceouspolyhedron envelope protein polyhedrin (Polh) (Friesen,1997; Rohrmann, 1992). In Autographa californica NPV(AcNPV), Polh is highly expressed during the verylate phase of infection and it must be transported intothe nucleus in large amounts, where occluded virions areassembled. Although Polh is produced abundantly ininfected pest cells, previous investigations have indicatedthat it is not an important factor for viral replicationand infection (Hu et al., 1999; Smith et al., 1983), but itinstead protects NPV virions from physical and biochem-ical inactivation in natural environments (Federici andHice, 1997; Rohrmann, 1986).

Escherichia coli is still the main expression system for theproduction of foreign proteins. Formation of insolubleinclusion bodies owing to overexpression in an E. coliexpression system can be a serious obstacle to foreignprotein expression due to the problem of refolding the

Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007 1183

Figure 1. Gene maps of recombinant plasmid pMPL104 and pMPL102. Plasmid

pTrcHisC was used as a parent vector. Abbreviations: Ptrc, trc promoter; polh, Polh

gene; gfpuv, UV-optimized GFP gene; RBS, ribosomal binding site; fp25, FP25 gene;

term, termination sequence; AmpR, ampicillin resistant gene; ColE1, replication origin;

lacIq, overexpressed Lac repressor.

protein into its native functional conformation (Rudolph,1995; Rudolph and Lilie, 1996). However, it also has severaladvantages, such as generally resulting in greater expres-sion of foreign protein (about 50% or more of the totalcellular protein under appropriate conditions), allowingeasy isolation of the protein at high purity (up to 90%purity under optimal conditions) and concentration,effectively protecting the target protein from proteolysis,and allowing efficient production of harmful or toxicproteins (Kane and Hartley, 1988; Lilie et al., 1998;Makrides, 1996). Previously, we showed that AcNPV Polhcould be successfully used as a fusion partner for theexpression of recombinant proteins (such as greenfluorescent protein (GFP), Bacillus thuringiensis (Bt) toxinprotein, and antimicrobial peptides) as insoluble inclusionbodies in an E. coli expression system, and that this Polh-fusion strategy enabled efficient production of the targetproteins (Seo et al., 2003, 2005; Wei et al., 2005). Inaddition, the recombinant Polh had almost the samecharacteristics as the native crystal matrix form ofbaculoviral polyhedra, including rapid solubilizationunder alkaline conditions and degradation by polyhedra-associated alkaline proteases. Therefore, using thesespecific properties of Polh protein, we successfullydemonstrated easy and efficient production and/orpurification of some target proteins (Seo et al., 2005;Wei et al., 2005).

fp25 gene is known to be involved in polyhedraformation, is located in AcNPV open-reading frame 61,and encodes the nucleocapsid structural protein FP25,which has a molecular weight of about 25 kDa (Braunagelet al., 1999). Even though FP25 is generally recognizednot to be a component of occluded virions, disruption ofthe fp25 by a 290-bp insertion element was observedfewer occlusion bodies in AcNPV infected insect cells(Bull et al., 2003) and mutations in the fp25 region of theAcNPV genome results in a phenotype that producesfew polyhedra (<10 per cell) compared with wild-typeisolates (Braunagel et al., 1999; Harrison and Summers,1995; Jarvis et al., 1992). In addition, mutations in thefp25 gene can affect the expression levels of severalbaculoviral proteins, including a marked reduction inPolh synthesis (Harrison et al., 1996) and significantincrease in the synthesis of several structural viral proteinssuch as gp67, p39 and BV/ODV-E26 (Braunagel et al., 1999;Rosas-Acosta et al., 2001). These investigations revealedthat FP25 might have a pivotal role at least in modulatingPolh synthesis and virion nuclear localization in insectsystem.

In this study, we investigated the effects of co-expressionof baculoviral FP25 on the production of baculoviral Polh-induced inclusion-body-formed fusion proteins in an E. coliexpression system. We used GFP as a target recombinantprotein owing to the ease with which it can be visualizedfrom outside the cell and its wide use as a fluorescentreporter protein (Cha et al., 2000; Johnvesly et al., 2004; Liet al., 2004).

1184 Biotechnology and Bioengineering, Vol. 96, No. 6, April 15, 2007

Materials and Methods

Bacterial Strains

E. coli TOP10 [F- mcrA D(mrr-hsdRMS-mcrBC)F801acZ-DM15 DlacX74 deoR recA1 araD139 D(ara-leu)7697 galUgalK rpsL (Strr) endA1 nupG] (Invitrogen) was used forrecombinant plasmid construction. E. coli BL21 [F� ompThsdSB (rB

� mB�) gal dcm] was used as a host strain forexpressing recombinant proteins.

Plasmid Construction

Recombinant plasmid pMPL104 that can co-express Polh-GFP and FP25 was constructed (Fig. 1). Polymerase chainreaction (PCR) was performed to generate a 619 bp of fp25encoding fragment using the primers (forward: 50-AAC-CATGGATCAATTTGAACAGTTGATTAACG-30, reverse:50-CGAATTCTTAAATTAAATTTTGAAGCATTTTTTCG-30) from AcUW1.lacZ viral DNA (BD PharMingen) that is amodified AcNPV baculovirus genomic DNA. The amplifiedfragment was digested with NcoI and EcoRI, and theninserted into the same digested sites of the plasmid vectorpTrcHisC (Invitrogen) to generate pMPL103. This plasmidwas further used as a template for PCR amplification of aribosomal binding site together with fp25 encoding

DOI 10.1002/bit

sequences using the primers (forward: 50-TACTG-CAGGTGGGCACTCGACCGGAATTATCG-30, reverse: 50-CGAATTCTTAAATTAAATTTTGAAGCATTTTTTCG-30).A 711 bp of amplified fragment was generated and digestedwith PstI and EcoRI, and then inserted into the same digestedsites of the recombinant plasmid pMPL102 containing polh-gfp fusion gene (Seo et al., 2003) to generate finalrecombinant plasmid pMPL104 (6,490 bp). The nucleotidesequences of the inserted genes were verified by directsequencing. For confirmation experiments, recombinantplasmid pPAPF that can co-express Polh-Hal18 and FP25was constructed based on the recombinant plasmid pPAPcontaining polh-hal18 fusion gene (Wei et al., 2005).

Media and Culture Conditions

For strain construction, cells were grown in Luria–Bertani(LB) medium. The constructed transformant harboringthe plasmid was stored at �808C. All cultures used formeasuring physiological characteristics were performed inM9 media (12.8 g/L Na2HPO4.7H2O, 3 g/L KH2PO4, 0.5 g/LNaCl, 1 g/L NH4Cl, 3 mg/L CaCl2, 1 mMMgSO4) with 0.5%(wt/vol) glucose as a sole carbon source in Erlenmeyer flasks,in an air shaking incubator with a gyration rate of 250 rpm.One milliliter of freezer stock was grown overnight (�12 h)at 378C in 10 mL LB medium in a 50-mL conical tube. Seedculture was transferred (5% vol/vol) to a final workingvolume of 50 mL in a 250-mL flask. Ampicillin (50 mg/mL)(Sigma) was added as a selection pressure for plasmid-harboring strains. Cell growth was monitored by measuringthe optical density at 600 nm (OD600) using a UV/VISspectrophotometer (Shimadzu). When cultures reached anOD600 of 0.4, 0.8 mM (final concentration) isopropyl-b-D-thiogalactopyranoside (IPTG; Sigma) was added to theculture broth for induction of recombinant protein. AfterIPTG induction, cells were grown at 258C. GFP fluorescenceintensity was determined using a fluorescence spectro-photometer (Shimadzu) at an excitation of 395 nm andemission of 509 nm.

Inclusion Body Isolation

The cell pellets from 100 mL cell cultures grown for 16 hwere resuspended in N-2-hydroxyethylpiperazine-N0-2-thanesulfonic acid (HEPES) buffer (50 mM HEPES-NaOHpH 7.5, 0.5 M NaCl, 1 mM phenylmethylsulfonylfluoride[PMSF], 5 mM dithiothreitol [DTT]) containing 0.35 mg/mL lysozyme (Sigma) at the ratio of 25 mLHEPES buffer for5 g wet cells, and then incubated at room temperature for30 min. Triton X-100 was added to a concentration of 1%(vol/vol), and the samples underwent ten 30-s bursts ofultrasound sonication treatment. Samples were collectedby centrifugation at 13,000 rpm for 30 min at 48C, and thenwashed twice with phosphate-buffered saline (PBS) buffer(pH 7) containing Triton X-100. The pellet fromcentrifugation at 13,000 rpm for 30 min at 48C was

regarded as purified inclusion bodies and stored at �208Cuntil use.

SDS–PAGE Analysis

Culture volumes equivalent to 1 mL were taken andcentrifuged at 13,000 rpm for 5 min at 48C. The cell pelletswere resuspended in Laemmli buffer (20 mM Tris �HCl(pH6.8), 2% (vol/vol) b-mercaptoethanol, 1 mM ethyle-nediamine tetraacetate (EDTA), 1% sodium dodecyl sulfate(SDS)) and boiled to 1008C for 5 min. After centrifugationfor 1 min, the samples were loaded onto a 12.5% (wt/vol)SDS–polyacrylamide gel for electrophoresis (SDS–PAGE).The gel was stained by 0.25% (wt/vol) Coomassie brilliantblue R-250 (Bio-Rad) for 30 min and destained by 45% (vol/vol) isopropanol and 10% (vol/vol) acetic acid. The gel wasscanned and its image was analyzed by Gel-Pro Analyzersoftware (Media Cybernetics).

Pronase Proteolytic Degradation and AlkalineSolubilization Analyses

The purified inclusion bodies were resuspended in PBS(pH 7) and then adjusted to give an optical density at 600 nm(OD600) of 1.0. Pronase (�4 units/mg; Sigma) was addedto a final concentration of 0.4 mg/mL. Protein suspensionswere incubated at 378C and GFP fluorescence intensities ofpronase-treated samples were measured at each 1-h interval.For solubilization analysis of inclusion bodies under alkalineconditions, the purified inclusion bodies were resuspended inan equal volume of PBS (pH 7 or 12). Protein suspensionswere then incubated at 378C and optical densities ofsolubilized samples were measured at each 1-h interval.

Electron Microscopy Analysis

Equal aliquots of the two cell suspensions (Polh-GFP andco-expressed Polh-GFP and FP25) were mixed with 2.5%glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h at 48C for theprimary fixation, then washed for at least 2 h at 48C in threechanges of 0.1 M PBS (pH 7.4) before 1% osmium tetroxidein 0.1 M PBS was added and the samples were incubated atroom temperature for 1 h for the secondary fixation. Afterremoval of fixative, the samples were rinsed in 0.1 M PBSthree times, then dehydrated with 50%, 70%, 90%, and 95%ethanol (15 min for each concentration), and four changesof 100% ethanol (15 min for each). After dehydration, thesamples were embedded in Spurr resin (Sigma) anddesiccated at 608C for 24 h, after which they underwentultrathin sectioning followed by staining with 5% uranylacetate and Reynold0s lead citrate solution (1.33 g leadnitrate, 1.76 g sodium citrate, 5 mL 1 N NaOH, distilledwater to 50 mL). Prepared specimens were examined usinga transmission electron microscope (TEM, H-7000FA;Hitachi).

Li et al.: FP25 Effect on Polh-Incusion Body in E. coli 1185

Biotechnology and Bioengineering. DOI 10.1002/bit

Figure 2. Time profiles of (A) cell growth and (B) specific GFP fluorescence

intensity in the culture of E. coli BL21 stains expressing (*) single Polh-GFP and (*)

Polh-GFP and FP25. Recombinant cells were cultured in 100 mL of M9 media with 0.5%

(wt/vol) glucose in 250-mL flasks at 250 rpm. When cultures reached an OD600 of 0.4,

0.8 mM IPTG was added to the culture broth for induction of recombinant proteins.

After IPTG induction, cells were grown at 258C. Each value and error bar represents

the mean of three independent experiments and its standard deviation.

Figure 3. Coomasie blue-stained SDS–PAGE analyses for checking (A) expres-

sion status and (B) purified inclusion body of sole Polh-GFP and Polh-GFP and FP25

and (C) expression status and (D) purified inclusion body of sole Polh-Hal18 and Polh-

Hal18 and FP25. Lane M: protein molecular weight marker; lane WC: whole cells; lane

S: soluble supernatant after sonication; lane IS: insoluble cell debris after sonication;

laneW/O: crudely isolated inclusion body without FP25 co-expression; laneW: crudely

isolated inclusion body with FP25 co-expression.

Results and Discussion

Co-Expression of Polh-GFP and FP25

Cell growth profiles showed that co-expressed additionalFP25 did not affect cell growth (Fig. 2A). As shown byultraviolet (UV) illumination (Fig. 2B, upper-right image)or fluorescence microscopic observation (data not shown),recombinant E. coli BL21 cells expressing Polh-GFP werefluorescent, demonstrating that some GFP molecules retainfunctionality in inclusion-body-formed fusion proteins.Interestingly, when expressed with FP25, Polh-GFP fusionprotein had markedly reduced visible GFP fluorescencecompared with Polh-GFP fusion molecules expressedalone (Fig. 2, lower-right image). These observations wereconfirmed by GFP fluorescence intensity measurements, inwhich Polh-GFP co-expressed with FP25 was found to havea 2.3-fold reduction in fluorescence intensity compared withPolh-GFP expressed alone (Fig. 2, left plot). It should benoted that a recombinant plasmid expressing mainly solublesingle GFP as a protein product, and which is a derivativeplasmid from the same parent vector pTrcHisC, had greater


GFP fluorescence intensity (more than 30-fold that of Polh-GFP) when grown under the same culture conditions (datanot shown). Thus, we surmised that cellular co-expressionof baculoviral FP25 might involve in inclusion-bodyformation by a more compact morphology and thus leadto a marked reduction in GFP fluorescence.

SDS–PAGE analysis was performed for whole cells,soluble supernatants, and insoluble cell debris after celldisruption to compare the patterns and quantities ofPolh-GFP fusion proteins expressed alone or with FP25co-expression (Fig. 3A). All samples except the solublesupernatants contained a protein with a molecular weight ofabout 58 kDa, which is inline with the predicted molecularweight of Polh-GFP fusion protein, indicating that theseproteins are expressed as insoluble inclusion bodies. ByWestern blot analysis with anti-GFP antibody, we confirmedthat these 58-kDa bands are Polh-GFP fusion proteins (datanot shown). Furthermore, a protein with a molecular weightof �25 kDa was present in the samples containing co-expressed Polh-GFP and FP25 (lanes WC and IS in Fig. 3A).Although we could not perform a Western blot to identitythis protein due to a lack of available anti-FP25 antibody, we

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concluded that it is likely to be FP25 as it was not present insamples of Polh-GFP expressed alone and its size accuratelycorrelated to the predicted molecular weight of FP25.Interestingly, FP25 was also absent from the solublesupernatant fraction. Because we isolated inclusion bodiesthat were mainly comprised of two proteins, Polh-GFP andFP25, from the SDS–PAGE results (Fig. 3B), we can surmisethat these two proteins are associated through their ownincorporation, and that FP25 might be involved in theformation of inclusion bodies with Polh-GFP fusionproteins. This result is in general agreement with theprevious report that FP25 protein is associated withocclusion derived virus (Braunagel et al., 2003). Interest-ingly, in the case of FP25 co-expression, the total amount ofcellular Polh-GFP was about six-fold greater than thatobtained when Polh-GFP was expressed alone (Fig. 3A, eachlane WC). Therefore, although visible GFP fluorescencefrom the E. coli cells co-expressing Polh-GFP and FP25 wasquite low compared with the strains expressing Polh-GFPonly (Fig. 2), the level of Polh-GFP expression was markedlyincreased with FP25 co-expression, suggesting that GFPfluorescence might be masked by compact morphology ofinclusion body with FP25 co-expression. We confirmed thatproduction levels of other Polh-fusion protein, antimicro-bial peptide Hal18 (Wei et al., 2005), was also enhanced(approximately two-fold) by FP25 co-expression (Fig. 3C)and FP25 might be involved in the formation of inclusionbodies (Fig. 3D).

Figure 4. Pronase proteolysis analyses on isolated inclusion bodies of

(*) single Polh-GFP and (*) Polh-GFP and FP25 that are resuspended in (A) normal

PBS buffer with pH 7 and (B) alkaline PBS buffer with pH 12. Inclusion bodies were

isolated using 16 h-cultured E. coli cells after induction. Relative value was based

on GFP fluorescence intensity at the initial incubation time. Each value and error

bar represents the mean of two independent experiments and its standard deviation.

Analyses for FP25 Effects on Polh-InducedInclusion Body Formation

As GFP is widely known to be resistant to many commonproteases except pronase (Bokman and Ward, 1981),pronase assays can be used to analyze the proteolyticdegradation of GFP. We performed pronase proteolysisanalyses to investigate the effects of FP25 co-expression onthe production and/or morphology of Polh-inducedinclusion bodies (Fig. 4). In the case of pronase digestionat neutral pH 7, the reduction in GFP fluorescence intensitywas smaller with co-expression of Polh-GFP and FP25 thanwith expression of Polh-GFP alone (Fig. 4A). Therefore, wecan surmise that pronase digestion reaction was limited onthe surface of Polh-GFP and FP25 inclusion bodies byincreased compact morphology of FP25-associated inclu-sion bodies. Because insoluble Polh or fused Polh can beeasily dissolved under alkaline conditions (Lavallee et al.,1993; Seo et al., 2003), we also investigated pronase digestionat pH 12 and found no significant difference in thereductions in GFP fluorescent intensities between the twoinclusion-body samples (Fig. 4B), demonstrating that bothsamples were dissolved at pH 12 and, therefore, that pronasecan digest GFP molecules of both samples with similarefficiencies.

In addition, we compared the solubilization properties ofFP25-associated inclusion bodies under neutral and alkaline

conditions with those containing Polh-GFP only (Fig. 5A).When resuspended in neutral PBS buffer (pH 7), the opticaldensity of each inclusion-body sample was unchanged.However, under alkaline conditions (pH 12), markedreductions in optical densities were observed for bothsamples, as expected. FP25-containing inclusion bodies hadhigher optical densities, whichmight indicate dissociation ofFP25 molecules from solubilized Polh-GFP fusion proteinsand/or compact morphology of FP25-containing inclusionbodies. However, through SDS–PAGE analysis for solubleand insoluble fractions after solubilization of Polh-GFP andFP25 inclusion body under alkaline condition (pH 12), weobserved that FP25 was also dissolved (Fig. 5B). That is,solubilization of Polh fusion induced co-solubilization ofFP25 protein and this indicate association of both proteins isnot just simple incorporation. Further investigation will beneeded for the structural and physiological relevance of Polhand FP25 proteins.



Figure 5. A: Optical density measurement analyses for solubilization of isolated

inclusion bodies of single Polh-GFP (open symbol) and Polh-GFP and FP25 (closed

symbol) under normal pH 7 (rectangular) and alkaline pH 12 (circle). B: Coomasie blue-

stained SDS–PAGE analysis for solubilization of Polh-GFP and FP25 inclusion body.

Lane M: protein molecular weight marker; lane S: soluble fraction after incubation in

PBS buffer; lane IS: insoluble fraction after incubation in PBS buffer. Inclusion bodies

were isolated using 16 h-cultured E. coli cells after induction.

Figure 6. Optical fluorescence microscopic images for (A) wild type cells,

(C) single Polh-GFP expressing cells, and (E) Polh-GFP and FP25 co-expressing cells

and transmission electron microscopic images for (B) wild type cells, (D) single

Polh-GFP expressing cells, and (F) Polh-GFP and FP25 co-expressing cells. Arrows

indicate the position of inclusion body. Bar scales: A, 6 mm; B, 250 nm; C, 5 mm; D,

550 nm; E, 6.8 mm; F, 550 nm.

To further investigate inclusion-body formation in E. colicells, TEM analysis was performed to directly observeinclusion bodies (Fig. 6). Interestingly, inclusion bodies inthe cells co-expressing Polh-GFP and FP25 had markedlygreater electron densities and larger sizes than those of thecells expressing only Polh-GFP (Fig. 6F vs. D), confirmingthat FP25 co-expression leads to high expression levels andcompact formation of Polh-induced inclusion bodies.

Expression of heterologous protein in E. coli often resultsin the formation of insoluble aggregates known as inclusionbodies (Fahnert et al., 2004; Villaverde and Carrio, 2003).The extent of protein aggregation is determined bymacromolecule concentration, domain numbers, environ-mental stress (especially, growth temperature), and avail-ability of molecular chaperones and folding catalysts(Baneyx and Mujacic, 2004; Jevsevar et al., 2005; Venturaand Villaverde, 2006). Although still a matter of argument,formation of inclusion bodies can be advantageous since


highly enriched proteins can be expressed and these proteinsare for the most part protected from proteolytic degradation(De Bernardez Clark, 1998, 2001). We have previouslyshowed that the fusion with baculoviral Polh conferred thehost cells to have higher heterologous protein yield (Seoet al., 2003, 2005; Wei et al., 2005). The results presentedhere demonstrated that the co-expression of baculoviralFP25 is able to modulate the accumulation of aggregation-prone recombinant fusion. Therefore, Polh and FP25 appearto be likely more advantageous partners for production offoreign proteins in E. coli system.

Although no investigations of FP25 co-expression effectson Polh expression have been reported using E. coli cells, theresults reported here are in general agreement with previouswork assessing the effect of fp25 deletions or mutations in aninsect expression system (Braunagel et al., 1999; Fraser et al.,1983; Jarvis et al., 1992). It was reported that mutations infp25 altered the level of Polh expression at the transcrip-tional level in insect cells, specifically by reducing the rate oftranscription (Harrison and Summers, 1995). However, thisreport was not fully supported, in that computer-assistedanalyses of the primary structure of FP25 did not indicatethe presence of RNA-binding motifs or any significant

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homology between FP25 and known transcriptional ortranslational regulatory proteins. Instead, FP25 may interactwith transcriptional and/or translational regulatory proteins(Rosas-Acosta et al., 2001). As this study in E. coli cellsshowed a direct increase on Polh levels with co-expression ofFP25, we therefore surmise that FP25 might functionthrough post-transcriptional or translational, rather thantranscriptional, mechanisms.

It has been conventionally believed that inclusion bodyproteins are biologically inactive, and therefore disregardedfor commercialization. However, structural characterizationstudies have shown that the protein aggregation is not an all-or-nothing process, but exhibits a continuum of foldingstates in both soluble and insoluble cell fractions (Schrodeland de Marco, 2005; Ventura and Villaverde, 2006). Severalreports have noted that some aggregation-prone enzymesexhibited biological activities (Garcıa-Fruitos et al., 2005;Tokatlidis et al., 1991). In the present study, althoughaggregated as inclusion body, either Polh-GFP or Polh-GFPand FP25 proteins were still fluorescent and this is consistentwith the observation by Garcıa-Fruitos et al. (2005); for thecase of GFP fused with a capsid protein of foot-and-mouthdisease virus, formation of inclusion body did notsignificantly inactivate the activity of GFP. Interestingly,although the amount of fusion protein in the presence ofFP25 was much higher than in the absence of FP25 (Figs. 3and 6), cellular fluorescence of the culture with FP25 co-expression was significantly reduced (Fig. 2). Here, we stressthat this is not apparently caused by the gene dosage butprobably by the architectonic nature of this kind ofaggregate because Polh might undergo itself assembly toform an aggregate prone fusion and FP25 associatesthrough intermolecular interaction which shielded thevisible GFP fluorescence. In fact, under alkaline condition,Polh-GFP can be easily dissolved without significant lossof GFP fluorescence (Seo et al., 2003) and FP25 was alsoco-dissolved (Fig. 5B). Because it was reported that theAcNPV Polh protein contains domains responsible forsupermolecular assembly (Jarvis et al., 1991), we surmisethat those domains in the Polh-GFP fusion might be alsofunctional in E. coli. Many studies on structural architectureof bacterial inclusion bodies have revealed that the enrichedintermolecular b-sheet structures are formed from thetested model proteins (Ami et al., 2003, 2005, 2006; Carrioet al., 2005; Garcıa-Fruitos et al., 2005) and thus, it will be ofinterest to further investigate the structure and function ofPolh and FP25-associated inclusion bodies.

Conclusions

In the present study, we found that co-expressed FP25 wasassociated with Polh-induced inclusion bodies in an E. coliexpression system and that its co-expression led to increasedproduction of fusion proteins. Therefore, a FP25 co-expression strategy can be used promisingly with a Polhfusion strategy to increase inclusion-body production

of foreign proteins, especially harmful proteins, in E. colicells.

The authors acknowledge support for fulfillment of this work by the

Marine Bioprocess Research Center of the Marine Bio 21 program

issued by the Ministry of Maritime Affairs and Fisheries, Korea, the

Brain Korea 21 program issued by the Ministry of Education, Korea,

and the Returned Overseas Chinese Scholars Program issued by the

Scientific Research Foundation, State Education Ministry, China.

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DOI 10.1002/bit

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