怎么去除胶水
西安电子科技大学研究生院
Journal of Biotechnology 123(2006)
236–247
Expression,purification and characterization of human GM-CSF
using silkworm pupae (Bombyx mori )as a bioreactor
Jian Chen a ,b ,Xiang-Fu Wu c ,Yao-Zhou Zhang b ,∗
a
College of Life Sciences,Zhejiang University,Hangzhou 310029,China b
Institute of Biochemistry,Zhejiang Sci-Tech University,Hangzhou 310018,China
c
Shanghai Institute of Biochemistry and Cell Biology,Chine Academy of Sciences,Shanghai 200031,China
Received 11June 2005;received in revid form 9November 2005;accepted 23November 2005
Abstract
To date,many recombinant proteins have been expresd in Bombyx mori cells or silkworm larvae,apart from in pupae.Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor.If maintained at an appropriate temperature,silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest.In this study,human granulocyte-macrophage colony-stimulating factor was successfully expresd in silkworm pupae i nucleopolyhedrovirus,purified and characterized with respect to its physico-chemical properties.The target protein expresd had an apparent molecular mass of 29kDa and an isoelectric point of 5.1.The protein was purified using three chromatographic steps with a final recovery of 10.3%.Finally,approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4×106cfu mg −1.The results of this study suggest that silkworm pupae re
prent a convenient and low-cost bioreactor for the expression of heterologous proteins.©2005Elvier B.V .All rights rerved.
Keywords:Human granulocyte-macrophage colony-stimulating factor;Bombyx mori nucleopolyhedrovirus;Silkworm pupae;Expression;Purification;Bioreactor
1.Introduction
心电图怎么看Mature human granulocyte-macrophage colony-stimulating factor (hGM-CSF)is an acidic glycopro-tein of 127amino acid residues,with a molecular mass ranging from 14.4to 32kDa (on SDS-PAGE),
∗
Corresponding author.Tel.:+8657186843194;fax:+8657186843198.
E-mail address: (Y .-Z.Zhang).
depending on the degree of glycosylation.This pro-tein acts on bone-marrow precursor cells and regulates proliferation and differentiation of gametocyte and monocyte/macrophage in vivo (Gasson,1991).Clinical studies have suggested that hGM-CSF is beneficial in treating myelodysplast
ic syndromes (Vadhan-Raj et al.,1987),marrow dia caud by cytotoxic chemother-apy (Antman et al.,1988),regenerative drawback ane-mia (Hord et al.,1995),visceral leismaniasis (Badaro et al.,1994)and AIDS (Hardy et al.,1994).At prent,
0168-1656/$–e front matter ©2005Elvier B.V .All rights rerved.doi:10.1016/j.jbiotec.2005.11.015
J.Chen et al./Journal of Biotechnology123(2006)236–247237
recombinant hGM-CSF(rhGM-CSF)for clinical appli-cation is mainly expresd in Escherichia coli;how-ever,this hGM-CSF is incorrectly folded and lacks post-translational modifications.
As Bombyx mori is susceptible to nuclear polyhe-drosis virus infection,Maeda et al.(1985)utilized i nucleopolyhedrovirus(BmNPV)system as an innovative tool for human␣-interferon expression.One of the major advantages of the BmNPV system is that it can be ud to produce relatively large quantities of post-translationally modified heterologous proteins in silkworm larvae.In addition,using silkworm lar-vae as a manufacturing factory or carrier,the BmNPV system has the following advantages compared to tho of the other baculovirus systems using Auto-grapha californica nucleopolyhedrovirus(AcNPV): higher yield,lower cost,relatively high abundance of post-tr
anslational modifications(Maeda,1989)and more stable products creted in hemolymph(Wu et al.,1990).However,silkworm larvae need to be reared with mulberry leaves or artificial diet and maintained under stable temperature(25–28◦C)conditions,which limit the systems that can be ud.Alternatively,silk-worm pupae may be chon to express the protein of interest using BmNPV.
回家的诗句In the prent study,we constructed a recombi-nant baculovirus vBm-CSF containing the hGM-CSF gene without the N-terminal signal peptide-encoded quence.Using this recombinant virus,we success-fully expresd hGM-CSF in silkworm pupae and puri-fied the protein using three chromatographic steps.It was found that the purified protein was glycosylated in the form of manno-type sugar residues and sig-nificantly different from that previously expresd in silkworm larvae(Shi et al.,1995,1996).However,the recombinant hGM-CSF expresd in silkworm pupae displayed the same biological activity as native hGM-CSF.The results indicate that silkworm pupae poten-tially reprent a more convenient and economical bioreactor for expression of heterologous proteins. 2.Materials and methods
2.1.Cell line,silkworm pupae,virus and culture conditions
The BmN cell line(maintained in our laboratory), derived from the i)ovary cell line,was
cultured in TC-100medium(Sigma)supplemented with10%(v/v)fetal calf rum(FCS,Gibco BRL) at27◦C.Silkworm i hybrid strain Qing-song×Haoyue)were commercially available and were maintained at2–8◦C before infection.The wild-type (WT)BmNPV(maintained in our laboratory)was propagated in BmN cells.
2.2.Cloning of hGM-CSF gene and construction
of a recombinant vector
One-step RT-PCR(Access RT-PCR kit,Promega) was performed to clone the hGM-CSF gene from total RNA,which was prepared from human embryofibrob-last tissue with Trizol reagent(Invitrogen).For RT-PCR,the following specific primers were designed: n,5 -GGGATATCATGGCACCCGCCCGCTCG-CCCAG-3 ;antin,5 -GGGGATCCTCACTCC-TGGACTGGCTCCC-3 .An Eco RV site and a Bam HI site(underlined)were introduced before the start codon and after the stop codon,respectively.The PCR reac-tion was performed for30cycles with three repeated steps:denaturation at94◦C for1min,annealing at 52◦C for1min and extension at68◦C for1min. The PCR product was gel-purified,digested by dou-ble restriction enzyme,and inrted into a BmNPV transfer vector named pUBM-4.This vector,similar to pBacPAK8(Clontech),is a recombinat
ion prod-uct of the vector pUC-19and an approximately7-kb fragment from the BmNPV genome,from which the polyhedrin gene was deleted but its promoter was remained.Briefly,the ligation products(named pBM-CSF)were transformed li competent TG1 cells(maintained in our laboratory)for screening pur-pos.A positive colony with the hGM-CSF gene in the plasmid was identified by double digestion of the plasmid,followed by analysis on1%agaro gel elec-trophoresis with subquent quencing(ABI Prism 3100-A).Finally,the recombinant pBM-CSF plasmid was isolated using a plasmid isolation kit(Roche)for subquent co-transfection.
2.3.Construction of vBm-CSF
BmN cells(approximately3×105)were eded into each well of a six-well plate(Falcon)in TC-100medium containing10%FCS.For transfection, after washing with rum-free TC-100medium,BmN
238J.Chen et al./Journal of Biotechnology123(2006)236–247
cells were maintained in2mL of rum-free TC-100 medium including100L of a mixture that contained 5L(approximately1g)of recombinant plasmid pBM-CSF,5L of Bsu36I-linearized BmNPV DNA (0.5g)and4L of Dosper(Roche).After incuba-tion for6h,cells was cultured in complet
e TC-100 medium containing10%FCS for72h at27◦C.The co-transfection supernatant was collected and10-fold dilutions were ud for plaque assays.According to the methods described by Summer and Smith(1987), three rounds of plaque screening for the isolation of pure recombinant virus were performed using dot blot hybridization.Thefinal virus yielding a maximum level of recombinant protein upon infection of silkworm BmN cells was titrated and propagated to obtain viral stocks.
To further confirm the recombinant virus,a primer was designed for single direct DNA quencing accord-ing to the BmNPV polyhedrin promoter quence as follows:5 -AACCATCTCGCAAATAAATAAGTA-TTTTACTGTTTTTCGT-3 .
2.4.Expression of hGM-CSF in silkworm pupae等高线地形图
Before infection,silkworm pupae were acclimatized to room temperature(25–28◦C).Pupae were subcu-taneously injected using a syringe(100L,Anting Factory,Shanghai)with10L of an amplified virus stock(106pfu mL−1)between the abdominal knobs on the backside.As a negative control,pupae were infected with a similar do of WT BmNPV.After infection,the pupae were incubated at room temper-ature and collected on thefifth day and then stored at−20◦C for subquent purification of the target protein.
2.5.Purification of rhGM-CSF from silkworm pupae
In this study,approximately500g of silkworm pupae infected with vBm-CSF was ud to extract the protein of interest.The pupae were homogenized in500mL of homogenization buffer[100mM sodium citrate,pH8.0,100mM KCl,2mM EDTA,2mM dithiothreitol(DTT),20g mL−1phenylmethyl-sulfonylfluoride(PMSF)]with a polytron PT2100 homogenizer(Kinematica,Luzern,Switzerland)at 20,000rpm.The homogenate was clarified by centrifu-gation at10,000×g for15min at4◦C.After removal of the lipid layer with a spatula,the supernatant was pooled and diluted with an equal volume of water.Solid (NH4)2SO4was slowly added to the diluted super-natant with constant mixing to achieve25%saturation, and the solution was then centrifuged at12,000×g for30min.The supernatant was adjusted to60% (NH4)2SO4and the precipitated proteins were pelleted. The resulting pellet was dissolved in100mL of buffer A(50mM Na-phosphate,pH7.0,1mM EDTA)and the solution was applied to a Sephacryl S-200gelfiltra-tion column(2.6cm×90cm,Amersham Biosciences) equilibrated with buffer A.Proteins were eluted with buffer A at aflow rate of1mL min−1.The active frac-tions were pooled and freeze-dried.The lyophilized sample was dissolved and then dialyzed against buffer B(50mM Tris–Cl,pH8.0,and1mM EDTA).The sample solution was fractionated on a HiPrep16/10Q FF(Amersham Biosciences)column equilibrated with buffer B.After a washing step wit
h5column volumes, bound proteins were eluted at4mL min−1with a 0–1M NaCl linear gradient.Active fractions eluted from the HiPrep Q column were lyophilized and then dialyzed against buffer B again.Thefinal purification of rhGM-CSF was performed using a Mono Q HR16/10column(Amersham Biosciences).The sam-ple was loaded onto the Mono Q column equilibrated with buffer B.The bound proteins were then eluted at2mL min−1with100mL of a0–1M NaCl linear gradient after a washing step with10column volumes. Active fractions were pooled and desalted on a HiPrep 26/10desalting column(Amersham Biosciences) with50mM Na-phosphate buffer,pH7.4.Thefinal products of purification were freeze-dried for further analysis.
2.6.Protein analysis
The amount of protein from each purification step was determined with a Micro BCA Protein Assay kit (Pierce).SDS-PAGE and Western blotting were car-ried out using the buffer system of Laemmli(1970), and proteins were visualized by Coomassie brilliant blue R-250staining.Isoelectric focusing(IEF)was performed using an Amersham Biosciences Phasys-tem according to the method of Righetti and Drysdale (1974).The N-terminal quence was determined by Edman degradation on an Applied Biosystem470A.
J.Chen et al./Journal of Biotechnology123(2006)236–247239
Deglycosylation analysis was performed according to the method described by Moonen et al.(1987),which was followed by SDS-PAGE on12%polyacrylamide slab gels with silver staining.Peptide mapping was achieved by reverd-pha HPLC according to the method described by Swiderek et al.(1999).
2.7.Immunoassay and biological assay
The protein of interest expresd in pupae was detected and quantified using a Quankine hGM-CSF ELISA kit(R&D System,Inc.)according to the manufacturer’s instructions.The primary antibody (goat anti-human GM-CSF antibody,R&D System, Inc.)and condary antibody(HRP-labeled rabbit anti-goat IgG,DAKOcytomation,Denmark)were ud for immunoblotting assay of the expresd products.
The bioassay of rhGM-CSF was performed with human bone-marrow cells(Metcalf,1984).Marrow mononuclear cells(MMCs)were parated over Ficoll-Conray(1.077)using density paration techniques (Boyum,1968).For granulocyte colony formation, an average of6×104MMCs were eded into25-mm Corning dishes containing10%FCS,0.4%low-melting agaro,rhGM-CSF and RPMI1640(Gibco BRL).The dishes were incubated at37◦C in a humidi-fied atmosphere of5%CO2for7days.Cell clusters containing40cells were scored as colonies.Units of colony-stimulatin
g activity were calculated from the linear portion of a do–respon curve,assigning 50U mL−1to the concentration stimulating the forma-tion of50%of maximal colony numbers(Okamoto et al.,1991).
3.Results
3.1.Amplification of hGM-CSF cDNA using
RT-PCR and construction of a recombinant vector As a result,an approximately400-bp DNA fragment was obtained using RT-PCR.After double restriction enzyme digestion,the amplified fragment was inrted into the baculovirus transfer vector pUBM-4to pro-duce recombinant transfer vector pBM-CSF,in which the5 and3 flank quences at two sides of the inrted fragment corresponded to AcMNPV and AcNPV in pBacPAK8,respectively,for recombination with WT BmNPV.DNA quencing analysis showed that the cloned fragment was identical to hGM-CSF gene with-out the signal peptide quence.This result indicates that the gene of interest was successfully cloned and inrted into the transfer vector.
3.2.Construction and identification of vBm-CSF
BmN cells were co-transfected with pBM-CSF DNA and Bsu36I-linearized BmNPV DNA.Recom-bina
nt virus vBm-CSF was obtained after veral rounds of plaque screening and purification,as iden-tified by dot blot hybridization.For quencing anal-ysis,viral DNA was prepared from the infected cells using alkaline lysis.DNA quencing indicated that the hGM-CSF gene had been transferred to the BmNPV genome.
3.3.Expression and identification of hGM-CSF in silkworm pupae
BmN cells at a density of2×106cells mL−1were infected with recombinant virus for5days at the fol-lowing titers:10,102,103,104,105,106,107,108, 109and1010pfu mL−1.Briefly,500L of BmN cells (approximately2×105cells)were eded into each well of a24-well plate and incubated at27◦C for 12–24h,when the cells adhered to the well bottom. The cells were then inoculated with5L of viral stock and were maintained for5days.Finally,BmN cell lysates were ud for quantitative analysis of recom-binant protein.The relationship between vBm-CSF titer and hGM-CSF expression showed that the expres-sion was optimal following infection with1L of 106pfu mL−1.
It was noted that3days after infection,the skin of silkworm pupae became dark,the inct bodies soft-ened and metamorphosis cead.In some cas,pupae died less than5days after infection.To analyze the amount of rhGM-CSF expresd in silkworm pupae, incts were randomly sampled at1
怀孕5个月胎儿图
2-h intervals after 48h of infection.The sampled pupae were homoge-nized and the homogenate was centrifuged16,000×g for10min at4◦C.After a dilution of1:10,000,the resulting supernatant of each sample was analyzed using a hGM-CSF ELISA kit and a standard curve. As a negative control,pupae were sampled24h after
孝敬父母手抄报240J.Chen et al./Journal of Biotechnology123(2006)
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Fig.1.Time cour of recombinant hGM-CSF expression in silk-worm pupae.After infection with virus,the incts were randomly sampled at an interval of12h.Due to the strong virulence of wild-type BmNPV,negative control pupae were sampled24h after infection.Silkworm pupae infected with vBm-CSF were sampled 48h after infection.The samples were analyzed with a hGM-CSF ELISA kit using a standard curve.
infection with WT BmNPV and analyzed in the same way.The results(Fig.1)showed that the production of recombinant protein incread continuously with increasing infection time.The amount of rhGM-CSF expresd in silkworm pupae was up to100g per pupa.
In addition,to identify the expression of rhGM-CSF, protein samples from silkworm pupae extracts(super-natant,kept at−20◦C)were parated by SDS-12% PAGE and Western blotting was carried out with a poly-clonal anti-hGM-CSF antibody(Fig.2).As shown in Fig.2,an approximately29-kDa protein band reacting against the poly-antibody was detected in samples from pupae infected with vBm-CSF.No immunoreactive protein was detected in the negative sample from the pupae infected with WT BmNPV.Thus,the results indicate that hGM-CSF was successfully expresd in silkworm工时制
pupae.Fig.2.Expression of hGM-CSF in silkworm pupae.M,protein mass markers;RV,extract of silkworm pupae infected with vBm-CSF; WT,extract of silkworm pupae infected with wild-type BmNPV. The arrow indicates the hybridized protein band.
3.4.Purification of hGM-CSF expresd in silkworm pupae
The recombinant protein expresd in silkworm pupae(named Bm-hGM-CSF)was purified by a ries of steps including ammonium sulfate precipitation,gel filtration on Sephacryl S-200and ion exchange chro-matography on HiPrep Q and Mono Q(Table1).In this work,silkworm pupae were chon as a resource for protein expression and purification.Thus,the whole pupa could act as a factory to produce the protein of
Table1
Purification profile of recombinant hGM-CSF expresd in silkworm pupae
Purification step V olume(mL)Protein(mg)Specific activity(g mL−1)Yield(%)Purification factor(fold) Homogenate170030511819.41001
(NH4)2SO4fractionation–91452–77 2.56
Sephacryl S-200180216884.24665
HiPrep Q20156296.718352
Mono Q4 3.5848.810.38979
