Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

PROTEIN EXPRESSION AND PURIFICATION ARTICLE NO.

7, 262–268 (1996)

0037

Expression of Human Growth Hormone in Silkworm Larvae through Recombinant Bombyx mori Nuclear Polyhedrosis Virus S. Sumathy, Vikas B. Palhan, and Karumathil P. Gopinathan1 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

Received September 12, 1995, and in revised form November 27, 1995

We have generated a recombinant Bombyx mori nuclear polyhedrosis virus, vBmhGH, harboring the fulllength human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 1 104 U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns. q 1996 Academic Press, Inc.

Growth hormone (GH) or somatotropin is a single chain polypeptide of about 22 kDa produced by the somatotrophs in the anterior portion of the pituitary gland. Proper synthesis and secretion of this hormone are essential for the normal growth and development of vertebrates. The human growth hormone (hGH) is a protein of immense biomedical importance and a number of strategies have been employed for its expression through recombinant DNA methodologies (1,2). 1

To whom correspondence should be addressed.

hGH being a nonglycosylated protein, prokaryotic expression systems have been exploited, using a variety of signal peptides to achieve export of soluble hGH to the periplasm (3–5). Prokaryotic expression systems, however, have limited value since the bacteria lack the splicing machinary if the introduced foreign gene is a genomic DNA harboring introns. The rapid adaptation and widespread use of baculovirus expression vectors for the production of heterologous proteins in insect cells have demonstrated the power of this emerging technology when the technical limits of traditional bacterial, yeast, or mammalian expression systems are reached (6,7). A key feature of the baculovirus based vectors is the utilization of a powerful promoter element which controls the expression of a gene encoding the major late viral protein, polyhedrin, the predominant constituent of the characteristic occlusion bodies (polyhedra) that accumulate in the nuclei of the infected cells during the late stages of infection. Foreign genes placed under the control of the polyhedrin promoter are transcribed at high rates by insect cells infected with the corresponding recombinant viruses, and large quantities of the encoded polypeptides are synthesized toward the end of the infection cycle. The prototype baculovirus used for expression of cloned foreign genes is the Autographa californica nuclear polyhedrosis virus and the insect host cell lines Sf9 or Sf21 (derived from Spodoptera frugiperda). An alternate baculovirus system with significant potential is the Bombyx mori nuclear polyhedrosis virus (BmNPV) based expression in BmN or Bm5 cells (derived from B. mori) (8). A major disadvantage of this system has been the low efficiency of transfection of cultured BmN or Bm5 cells and consequently the lower frequencies for recombinant virus generation. However, recently we have achieved a technical breakthrough for improved transfection efficiency in BmN cells through lipofection by which recombinant

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1046-5928/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BmNPV could be readily generated (9). Such recombinant BmNPV could be used to produce cloned proteins in large amounts in BmN cell lines or in B. mori larvae. Proteins synthesized in insect cells undergo a variety of intracellular post-translational modifications, and in many cases the modifications have resulted in the acquisition of biological activity (10). In general, the cDNA clones rather than genomic clones have been used for high level expressions through the baculovirus system. Thus far, intron containing genes such as the early transcription unit of simian virus 40 and silk moth chorion, each with one intron (11,12), and the immediate early protein 1 (IE1) of human cytomegalovirus with two introns (13) have been expressed in baculovirus systems. In this study we have explored the expression of a chromosomal gene, human growth hormone (hGH) harboring four introns as well as the signal peptide coding sequences through recombinant BmNPV, in BmN cells as well as in silkworm larvae. We demonstrate that insect cells infected with recombinant BmNPV are capable of transcribing the hGH driven by the polyhedrin promoter and synthesizing the encoded polypeptide. Further the correct post-transcriptional processing of the growth hormone transcripts occurs and the protein is processed post-translationally in the infected cells to yield the mature growth hormone, which is immunologically and biologically active. The recombinant hGH from larval hemolymph has been purified to near homogeneity in a single step immunoaffinity chromatography. MATERIALS AND METHODS

Cell lines. The B. mori-derived cell line BmN was received from Dr. Susumu Maeda, University of California, Davis. The cell line was maintained at 277C in TC 100 medium (GIBCO Laboratories) supplemented with 10% fetal calf serum. Construction of the recombinant plasmid transfer vector pBm030-hGH. All standard recombinant DNA protocols were followed as described (14,15). The hGH gene present in the plasmid pTKGH (Allegro, Nichols Institute Diagnostics, CA) contained the signal peptide sequences, the coding sequences of mature hGH, and the 3* noncoding sequences, including the signal for polyadenylation. The hGH gene was subcloned as a 2.4-kb EcoRI fragment from pTKGH, into the BmNPV polyhedrin based transfer vector pBm030 (16) at the EcoRI site. Generation of the hGH-recombinant B. mori nuclear polyhedrosis virus (vBmhGH). The transfer vector pBm030-hGH harboring the genomic copy of hGH under the control of the BmNPV polyhedrin promoter was cotransfected along with the genomic DNA of wild-type BmNPV into BmN cells (1 1 106 cells) using the im-

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proved lipofection technique (9). Six days after the transfection, the culture medium was removed and the recombinant virus was isolated by the end point dilution method in 96-well plates. The recombinant virus isolates, showing cytopathic effect but no polyhedral inclusion body production, were purified by plaque assay (16). The purified recombinant virus vBmhGH was propagated by infecting BmN cells on a large scale and was stored at 0707C. The titer of the recombinant virus preparation determined by the end point dilution method was about 107 PFU/ml. Analysis of RNA. Total cellular RNA was isolated from monolayer of BmN cells by the guanidinium isothiocyanate method (17). RNA was isolated from 4 1 106 cells, infected with recombinant vBmhGH or wildtype BmNPV. For Northern blot analysis, the total RNA was electrophoresed on 1.2% agarose formaldehyde gels and transferred to nylon membrane. The RNA was uv-crosslinked to the membrane and probed using a 32P-labeled hGH DNA probe. Metabolic labeling of cells and preparation of cell extracts. Uninfected and vBmhGH recombinant virusinfected BmN cells were left covered with TC100 medium lacking methionine and fetal calf serum for 1 h and labeled in the same medium for 2 h in the presence of 25 mCi of [35S]methionine. The culture medium was saved and the proteins were extracted from the cells. For protein extraction, the cells were rinsed at 47C with phosphate-buffered saline, 0.01 M sodium phosphate, pH 7.4, 0.14 M NaCl) followed by wash buffer (20 mM Tris–HCl, pH 7.5, 0.135 M NaCl, 1 mM MgCl2 , and 10% glycerol). The washed cells (2 1 106) were treated with 1 ml of lysis buffer [wash buffer plus 1% Nonidet P-40 (NP-40), 2 mM phenylmethylsulfonyl fluoride, and 10 ml of aprotinin/ml] per 60-mm dish for 20 min at 47C and the insoluble debris was removed by centrifugation. Immunoprecipitation and polyacrylamide gel electrophoresis. Cell extracts and culture supernatants were incubated with polyclonal antibody raised against purified hGH for 1 h at 47C. The incubation was continued for 30 min in the presence of 50 ml of a 50% suspension of protein A–agarose beads (Bangalore Genei, India). Following this, the beads were pelleted by centrifugation and washed twice with 0.01 M Tris–HCl, pH 8.0, containing 0.1 mM EDTA, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS. The beads were boiled in Laemmli standard sample buffer (18) for 5 min and the eluted proteins were analyzed by SDS– PAGE. Expression of hGH in silkworm larvae. B. mori larvae (Bivoltine race, NB4D2) were reared on mulberry leaves at 257C. About 0.5 1 105 PFU of the recombinant virus was inoculated subcutaneously into the body cavity of the larvae (fifth instar, second day) with a sy-

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ringe. Four to 5 days postinfection, the hemolymph was collected by piercing the abdominal legs and stored frozen at 0707C. The presence of hGH in the hemolymph was analysed by SDS–PAGE and immunological assays. Western blotting. Protein samples (hemolymph) were electrophoresed through 10% SDS–polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 3% bovine serum albumin, in 10 mM Tris–HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20, for 1 h at 377C. The blot was rinsed with the same buffer lacking bovine serum albumin (TST buffer) and incubated with primary antibody (1:400 dilution in TST buffer) for 1 h at 227C. The blot was washed three times with TST buffer and incubated with a 1:500 dilution of anti-rabbit immunoglobulin G conjugated to horse radish peroxidase (Bangalore Genei, India) in TST buffer. The blots were monitored for color development in the presence of diaminobenzidene and hydrogen peroxide. Radioimmunoassay (RIA) and radioligand receptor assay (RRA). The RIA was performed using 125I-hGH and anti-hGH antibody (NHPP, Baltimore, MD) essentially as described (19). The iodination of hGH tracer was carried out with chloramine T method. The specific activity of the labeled probe was about 1 mCi/mg. 125IhGH (100,000 cpm) and varying concentrations of unlabeled hGH or recombinant hGH (rhGH) were mixed with anti-hGH antibody in 300 ml of RIA buffer (50 mM sodium phosphate buffer, pH 7.2). After incubation at 257C overnight, the bound tracer was precipitated with anti-rabbit IgG (1:3 dilution) plus 1:10 diluted normal rabbit serum. The whole complex was precipitated with 5% PEG 8000 and the radioactivity was determined in a Beckman gamma counter. The RRA was carried out using 125I-hGH and a relatively crude preparation of hGH receptor from rat liver homogenate (20). The mixture (300 ml) containing labeled 125I-hGH (100,000 cpm), 100 ng crude receptor, and varying concentrations of rhGH or hGH were incubated at 377C for 2 h. The reaction was stopped with cold RRA buffer (20 mM Tris–HCl, pH 7.0, 10 mM MgCl2 , 0.5% BSA, and 0.1% NaN3) followed by centrifugation at 4000 rpm for 30 min at 47C. The supernatant was discarded and the pellets were counted in a gamma counter. Purification of hGH expressed in silkworm. About 10 ml of hemolymph collected from 20 silkworm larvae infected with vBmhGH were pooled and dialysed against PBS, pH 7.2. After dialysis, the hemolymph was clarified by centrifugation at 12000 rpm for 5 min. The supernatant was applied onto a column of protein A–Sepharose to which the polyclonal antibody (rabbit) against hGH was bound. The column was washed several times using PBS, pH 7.2, containing 20% glycerol.

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FIG. 1. Construction of transfer vector pBm030-hGH. A 2.4-kb EcoRI fragment harboring the hGH from plasmid pTKGH (see Materials and Methods) was inserted into the EcoRI site of the transfer vector pBm030. The transfer vector has BmNPV polyhedrin promoter sequences (Ppolh) along with 2.9 kb 5* upstream and 4 kb 3* downstream polyhedrin-flanking sequences (indicated as light grey shaded areas, 5* FR and 3* FR, respectively) and unique BglII and EcoRI sites at the multiple cloning site. The insert fragment carries the chromosomal copy of hGH consisting of five exons (darkly shaded) and four introns (shaded grey), the 5* signal sequences, as well as the 3* noncoding sequences up to 526 bp past the polyadenylation signal. The sizes of the exons from 1 to 5 are 10, 161, 120, 165, and 198 bp, and the introns from one to four are 256, 209, 93, and 253 bp, respectively. The orientation of the hGH insert was confirmed by digestion with BglII.

The hGH bound to the affinity matrix was eluted using 100 mM glycine–HCl buffer, pH 2.5, containing 20% glycerol. The eluate was instantly neutralized with 1 M Tris–HCl, pH 8.8, and the individual samples were analyzed by SDS–PAGE. The fractions were also assayed for hGH by ELISA (GIBCO BRL). Protein concentrations were determined by Lowry’s method (21). RESULTS

Construction of transfer vector harboring hGH. The recombinant plasmid construct pBm030-hGH (Fig. 1) harboring the chromosomal copy of the human growth hormone gene was generated by inserting a 2.4-kb genomic fragment to the transfer vector pBm030. The insert contained the entire hGH structural gene sequences consisting of five exons and four introns and the 3* flanking sequences beyond the polyadenylation signal. The orientation of the hGH insert was confirmed by restriction with BglII. Generation of recombinant vBmhGH. A recombinant BmNPV harboring hGH was generated by recombination between plasmid pBm030-hGH DNA and wild-type BmNPV genome following lipofection in BmN cells. The recombinant virus, vBmhGH, was initially identified by the occlusion negative (Occ0) phenotypes. At least four independent Occ0 isolates obtained from 96-well screening were further purified by plaquing twice and propagated in BmN cells. Out of many plaques obtained from each of these four isolates, three

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FIG. 2. SDS–PAGE of proteins from vBmhGH-infected cells. BmN cells (2 1 106 cells) infected with wild-type BmNPV or recombinant vBmhGH were labeled for 1 h using [35S]methionine after 48 h post infection (for details see text). Culture supernatant from vBmhGHinfected BmN cells was removed after labeling and was immunoprecipitated using rabbit polyclonal antibodies against purified hGH. The labeled immunoprecipitated proteins were analyzed by 12% SDS–PAGE. Lanes: 1, BmN cells, mock infected; 2, cells infected with wild-type BmNPV; 3, cells infected with recombinant BmNPV (vBmhGH). The position of the immunoprecipitated polypeptide, hGH, is indicated by an arrow.

were finally selected and their identities were confirmed by dot hybridization. To scale up the recombinant virus, a monolayer of BmN cells was infected with the recombinant virus at a multiplicity of infection of 0.5. After 5 days, the presence of hGH in the culture medium and the cell lysate was monitored by metabolic labeling with [35S]methionine. The presence of hGH was clearly observed in the culture medium, although not to high levels (Fig. 2). The presence of hGH could not be detected in the cell pellets. Although insect cells infected with baculovirus are inefficient at secretion, the efficient secretion of hGH observed here could be due to the low production level. Analysis of hGH transcription. Since the levels of hGH protein secreted were low, the transcriptional level of recombinant hGH was examined. Figure 3 shows Northern blot analysis of mRNA expressed in BmN cells infected with the recombinant virus. The mature hGH transcript (0.8 kb) though discernible was less abundant than the precusor species, possibly due to the inefficient processing of the primary transcripts of hGH in these cells. The low level of biologically active hGH in BmN cells could thus be due to low efficiency of splicing. Expression of recombinant hGH in B. mori larvae. To see whether better yields of recombinant hGH can be achieved in B. mori larvae rather than in cell lines, we expressed the protein in silkworm larvae following the recombinant virus infection. A 22-kDa protein band

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FIG. 3. Northern blot of hGH mRNA synthesized by BmN cells. Twenty micrograms of the total RNA isolated from BmN cells (uninfected or virus infected at 48 h post infection) were resolved by electrophoresis through an agarose–formaldehyde gel and blotted onto a nylon filter. hGH mRNA was visualized by autoradiography after hybridization with a 32P-labeled hGH DNA probe (3 1 107 cpm) in 6 ml hybridization buffer containing 50% formamide and 51 SSC (11 SSC is 0.15 M NaCl and 0.015 M sodium citrate) overnight at 427C. The filter was washed for 30 min in 21 SSC–0.1% SDS at room temperature followed by 20 min in 0.11 SSC–0.1% SDS at 607C and then exposed to X-ray film. The size of the hGH mRNA was estimated relative to stained RNA markers in an adjacent lane. Lanes: 1, recombinant virus (vBmhGH) infected; 2, wild-type BmNPV infected; 3, uninfected BmN cells.

corresponding to the size of native hGH was observed in SDS–PAGE analysis of larval hemolymph (Fig. 4). This protein was clearly absent from the wild-type NPV-infected B. mori hemolymph. The identity of the

FIG. 4. Synthesis of hGH in silkworm larvae. B. mori larvae (bivoltine strain, NB4D2 race: 5th instar, 2nd day) was infected with wildtype or recombinant BmNPV. Four days following infection, the larvae were bled and the hemolymph was collected into a chilled microfuge tube containing a crystal of phenylthiourea to avoid melanization of hemolymph. Hemolymph samples were electrophoresed through 12% polyacrylamide gels under reducing conditions and the protein bands were silver stained. Lanes: 1, protein size markers (kDa); 2, standard hGH; 3 and 4, hemolymph from vBmhGH-infected and wild-type BmNPV-infected larvae, respectively.

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FIG. 5. Radioimmunoassay and radioligand receptor assay of rhGH. (A) For radioimmunoassay 0.01–1 pmol of rhGH (s) or hGH (l) was incubated with 100,000 cpm 125I-hGH and rabbit anti hGH serum (1:128,000) in 300 ml of 50 mM sodium phosphate buffer, pH 7.2, at 257C overnight. The bound tracer was separated by precipitation with PEG 8000 and the radioactivity was determined. (B) Radioligand receptor assay of rhGH. The receptor for GH was isolated from livers of pregnant rats. Briefly, the livers were minced and homogenized in ice-cold 0.25 M sucrose and centrifuged at 4000g for 20 min at 47C. The supernatant was diluted with equal volumes of 0.025 M Tris, pH 7.6, containing 20 mM CaCl2 and centrifuged at 15,000g for 20 min at 47C. The pellet comprising the membrane receptors for GH was resuspended in 50 mM Tris–HCl, pH 7.4, and used for the assay. Binding was performed by incubating approximately 100 ng of the crude GH receptors with 100,000 cpm of 125I-hGH in the presence of varying concentrations of either hGH (s) or rhGH (l) for 2 h at 377C. The bound 125I-hGH was measured as described in the text.

protein to hGH was confirmed by its specific reaction with anti-hGH antiserum in a Western blot (see Fig. 6A, lane 1). Individual silkworm larvae produced 20–50 mg of hGH following vBmhGH infection. This corresponds to an average yield of 40–100 mg of hGH per liter of hemolymph of the infected insects. Immunological properties of rhGH. The immunopotency of rhGH in B. mori hemolymph was determined by RIA using 125I-hGH and anti-hGH antibodies and compared to the native hGH. As shown in Fig. 5A, the rhGH and native hGH displayed close parallel dose– response curves, indicating the existence of common epitopes. The amounts of rhGH and native hGH to achieve 50% inhibition of tracer binding with antibodies were 0.8 and 0.7 pmol, respectively. Thus the immunopotency of rhGH was about 90% of that of the native molecule. In vitro biological properties of rhGH. The ability of rhGH to bind to the rat liver membrane receptors for growth hormone was determined by RRA (Fig. 5B). As evident from the figure, rhGH and native hGH yielded almost overlapping displacement curves. The amount of rhGH required for 50% inhibition of binding was 5.5 pmol as compared to a value of 3.5 pmol by native hGH, indicating the close similarity in the affinity and avidity of binding of rhGH and hGH to GH receptors.

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Purification of rhGH. The purification of rhGH from the hemolymph of larvae infected with vBmhGH was achieved using an immunoaffinity matrix to which rabbit anti-hGH antibodies were attached noncovalently. The individual fractions were assayed for the presence of hGH by ELISA. The fractions containing hGH were pooled and analyzed by SDS–PAGE, and a 22-kDa band corresponding to the native hGH was detected (Fig. 6B, compare lanes 1 and 2). This preparation showed a specific activity of 24,000 U/mg with 51% recovery and approximately 2666-fold purification (Table 1). The proteins from crude hemolymph and the immunoaffinity-purified samples (lanes 1 and 2 respectively in Fig. 6A) showed immunological reactivity comparable to authentic hGH (lane 3). Under the conditions employed for the elution of the rhGH from the immunoaffinity column, we did not see the presence of any free IgG in the eluate, for reasons not known. The final preparation of recombinant hGH was more than 95% pure as judged from the staining sensitivity of proteins in SDS–PAGE. DISCUSSION

The results presented here have shown that employing the baculovirus BmNPV system efficient expression of a foreign protein can be achieved from genomic, multiple-intron containing genes in BmN cells or B. mori larvae. Northern analysis of the hGH RNA

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RECOMBINANT hGH EXPRESSION IN SILKWORM LARVAE TABLE 1

Purification of Recombinant hGH

Purification step

Protein (mg)

Total activity (U)a

Yield (%)

Specific activity (U/mg protein)

Purification (fold)

Hemolymph Immunoaffinity column

260 0.05

2340 1200

100 51

9 24000

1 2666

Note. The crude hemolymph and the individual fractions were assayed for hGH by ELISA (GIBCO, BRL). Briefly, the samples were allowed to react with anti-hGH (monoclonal antibodies) coated to the plates. The resulting antigen–antibody complex was captured by rabbit antibodies to hGH. This complex was detected using anti-rabbit IgG conjugated to horse radish peroxidase. The reaction was developed using hydrogen peroxide and orthophenylenediamine and the A490 nm was monitored. The concentration of hGH in the samples was deduced from the standard graph generated using pure hGH. a One unit was arbitrarily defined as 0.1 A490 nm in ELISA.

transcripts accumulating in BmN cells infected with the recombinant BmNPV has established that even at the late stages of infection, infected cells maintain their basic ability to undertake correctly all the steps of the mRNA biosynthetic pathway, including splicing. We have observed that BmN cells infected for a period of 48 h or less contained more immunoprecipitable polypeptides than longer infections (data not shown). This observation seen in the light of incomplete processing of the primary transcripts reported here suggests that

FIG. 6. Purification of rhGH expressed in silk worm larvae. The hemolymph samples from vBmhGH-infected B. mori larvae (shown in lane 3, Fig. 5) loaded onto a column of protein A–Sepharose to which rabbit polyclonal antibodies to hGH were bound. The column was washed several times with PBS, pH 7.0, and the bound proteins were eluted (500-ml fractions) using 100 mM glycine buffer, pH 2.5. The presence of hGH in individual fractions was assayed by ELISA. The fractions containing hGH were pooled and dialysed. The clarified crude hemolymph and the purified protein samples (pooled fractions) were boiled in SDS loading buffer and analysed by 12% SDS–PAGE. (B) Silver staining of the proteins. Lanes: 1, 10 mg of standard hGH; 2, 10 ml (500 ng) of the pooled fractions eluted from immunoaffinity column; and 3, protein size markers (kDa). (A) Immunoblot analysis. The proteins after electrophoresis were transferred to nitrocellulose membrane, treated with rabbit antibodies to hGH and detected by the immunoperoxidase activity. Lanes: 1, hemolymph from recombinant vBmhGH-infected B. mori larvae (200 mg total protein corresponding to 600–700 ng hGH); 2, affinity-purified hGH (500 ng); 3, standard hGH (10 mg).

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the splicing machinery of the cells may be gradually incapacitated at the late stages of viral infection. A more systematic analysis is, however, needed for making a firm correlation between relative and absolute accumulation of correctly spliced transcripts as a function of the duration of infection. One advantage of the BmNPV expression system is the high level of foreign protein production in B. mori larvae ranging from 20- to 500-fold higher than that in established cell lines (22). This is largely influenced by the particular gene product synthesized. The hGH production in the present system was better in B. mori larvae than that in cell culture. The feasibility of obtaining authentic gene products from chromosomal genes placed under polyhedrin promoter control obviates the need for generating full length cDNA clones for protein identification purposes. The rhGH was found to be identical to the native hGH in various immunological and biological properties such as its ability to bind to the antibodies or to the receptors. All these properties taken together indicate that rhGH probably undergoes proper folding into the right conformation as present in the native hGH. In summary, the successful, albeit low, expression of hGH in insect cells under the control of the polyhedrin promoter suggests that the baculovirus system should be of general applicability and is efficient in processing of transcripts from complex chromosomal genes of higher eucaryotes. Our data provide the first report of an eukaryotic chromosomal gene being spliced and expressed under the control of the polyhedrin promoter using the BmNPV-based baculovirus system in cell lines as well as in the larval caterpillars. The expression level of rhGH (20–50 mg/larvae) achieved from the chromosomal gene may be further improved by using a cDNA hGH cassette. ACKNOWLEDGMENTS We thank the National Hormone and Pituitary Program, NIDDK, NICHHD, and USDA for the supply of RIA reagents for human

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growth hormone (Lot Nos. APPC119810, AFP11019B, AFP4793B). We also thank Drs. A. J. Rao and Rajan Dighe, Centre For Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, for the generous supply of pure hGH and rabbit antibodies against hGH and helpful advice in RIA and RRA. This work was supported by grants from the Department of Biotechnology, Government of India.

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