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Biosynthesis and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori
Materials Science and Engineering C 29 (2009) 1321–1325
Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Biosynthesis and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori ☆ Jian-Nan Wang a,⁎, Shu-Qin Yan a, Chang-De Lu b, Lun Bai a a b
College of Material Engineering, Soochow University, Suzhou 215021, PR China Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China
a r t i c l e
i n f o
Article history: Received 16 August 2007 Received in revised form 8 September 2008 Accepted 22 October 2008 Available online 5 November 2008 Keywords: Fibroin crystalline Silkworm Genes Fusion protein Thrombin ThT
a b s t r a c t We aimed to investigate the self-organization/self-assembly mechanisms of silkworm fibroin-based material. In the present study, for the first time, we designed and multimerized four DNA “monomer” sequences from structurally simple fibroin crystalline peptides or analog, [GAGAGX] (X = A, S, Y and V) to encode polypeptides [GAGAGX]16 (eGA, eGS, eGY and eGV) using a “head-to-tail” construction strategy. Multimers were cloned into pGEX-KG and fusion proteins GST-[GAGAGX]16 (KGA, KGS, KGY and KGV) were efficiently expressed in Escherichia coli. These fusion proteins were isolated and purified by GST affinity chromatography and confirmed by SDS–PAGE and Western blot analysis using antibody reactive to GST. The polypeptides were cleavaged from GST fusion proteins by digesting with thrombin enzyme. The composition of the four polypeptides was confirmed by composition analysis of amino acids, and their abilities to form βsheet structure were determined by ThT fluorescence spectral analysis. The content of β-sheet among the four polypeptides followed the order: eGS N eGV N eGY NeGA. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Silk fibroin of silkworm Bombyx mori is synthesized in the posterior silk gland cells of silkworm and secreted into the lumen. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility [1–5], slow degradability and remarkable mechanical properties of the material. An ideal medical material for tissue engineering should not only have good biological compatibility, but also possess appropriate porosity, and controlled structure and properties, especially controlled degradation [6]. Silk fibroin of B. mori is a crystal high polymer with three crystalline modifications, silkI, silkII and silkIII [7–13]. SilkII is the major formed part of stable β-sheet in natural silk fibroin [7,10] or regenerated silk fibroin [14,15], it is difficult for silk fibroin to be biodegraded [16,17]. Few studies focused on controlling the condensed structure of regenerated silk fibroin to improve degradation [18], but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes.
Abbreviations: Fib-H, silk fibroin heavy chain; Fib-L, silk fibroin light chain; ThT, thioflavin T; GST, glutathione transferase; G, Gly; A, Ala; S, Ser; Y, Tyr; V, Val; L, Leu; P, Pro; R, Arg. ☆ This work was completed at Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. ⁎ Corresponding author. Tel.: +86 512 67487067; fax: +86 512 67246786. E-mail address: [email protected] (J.-N. Wang). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.10.029
Silk fibroin of the silkworm B. mori is a kind of structural protein. Protein architecture and mechanical properties are determined by its amino acid composition and sequence. Change in molecular composition by gene engineering would result in desired structural features and varied properties [19], including degradation. Each molecule of silk fibroin consists of Fib-H and Fib-L, which are linked by a disulfide bond of the two subunits [20], and another glycoprotein fibrohexamerin/P25 (FHX/P25), which associates with the H–L complex by hydrophobic interaction at a ratio of 1:6:6 to form an elementary unit of silk fibroin [21–24]. Fib-H is a hydrophobic protein composed of alternate arrays of 12 crystalline domains (repetitive domains) and 11 amorphous domains (non-repetitive domains), and its complete gene sequence and amino acid composition were recently determined by Zhou et al. [25,26]. The regular amino acid sequence of Fib-H, a crystalline domain, is distributed into several subdomains beginning with a stretch of [GAGAGS] hexapeptides and ending in [GAAS] tetrapeptide. [GAGAGY], [GAGAGA] and [GAGAGV] hexapeptides are also distributed in the crystalline domain, with the copy numbers of four hexapeptides 433, 120, 27 and 30. The [GAGAGS] hexapeptide assumes a β-sheet conformation and plays an important role in formation of better crystalline domains [27]. But functions of Tyr and Val or other amino acids in fibroin molecule conformation and property are still unclear. It is of great interest to study the modification of silk fibroin using genetic engineering. In the present study, we described the preparation of the polypeptides [GAGAGX]n (X = A, S, Y and V) containing highly repeated consensus 6 amino acid motif [GAGAGS] from silk fibroin crystalline of silkworm or various analog, and characterized their abilities to form β-
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2. Experiments procedure
per milligram fusion protein was cleavaged with 1 U thrombin (Novagen) in 1× cleavage buffer at 20 °C for 16 h. The reaction mixture was diluted in GST binding buffer and loaded onto GST affinity column to remove GST-tag.
2.1. Synthetic gene assembly expression vector constructs
2.5. SDS–PAGE electrophoresis and Western blot analysis
The DNA monomers of 18 base pairs (Fig. 1, underlined) derived from Fib-H genes and encoding hexapeptides GAGAGX(X = A, S, Y and V) were designed and then polymerized to 16 repeats by repeated doubling in Escherichia coli (E. coli). Multimers were formed by insertion of ScaI/NgoMIV digested fragments into ScaI/AgeI digested vectors, these enzymes generate identical cohesive ends, and when the ligation joins a NgoMIV and a AgeI site together in a “head-to-tail” fashion, any internal restriction sites are destroyed. The extended genes, flanked by AgeI and HindIII sites, were inserted into the expression vector pGEX-KG-AgeI at the same sites following the gene sequence encoding GST. pGEX-KG-AgeI was derived from pGEX-KG by inserting synthetic double-stranded oligonucleotides (5’ GATCCACCGGTGG 3’, 5’ AATTCCACCGGTG 3’) fragment containing a AgeI site (underlined). The resulting plasmids were designated pGEX-KGA, pGEX-KGS, pGEX-KGY and pGEX-KGV. All cloned fragments were further verified by nucleotide sequencing (Invitrogen).
The protein content within the whole cell lysate, the purified fusion protein and the liberated polypeptide were identified on SDS– PAGE of 12% polyacrylamide gel (Sigma) by adding equal volume of sample buffer (20% 2-mercaptoethanol; 4% SDS; 20% glycerol; 0.2% bromophenol blue; 0.1 M Tris–HCl, pH 6.8) and boiling for 3–5 min, finally stained by Coomassie brilliant blue. After SDS–PAGE, the proteins (within the whole cell lysate) were transferred onto a PVDF membrane(Immobilon-P, Millipore), and subsequently blocked with TBST (50 mM Tris–HCl, pH 7.6; 150 mM NaCl; 0.05% Tween-20) containing 5% of nonfat dried milk. Then membrane was incubated in TBST containing 5% of nonfat dried milk and 1000× diluted GST antibody (Stressgen) for 1 h, after washing with TBST three times (each for 10 min), followed incubation of horseradish peroxidase labeled rabbit IgG antibody (Jingmei Biotech Co., Ltd.). The antibody was detected using DAB reagent (Shanghai Changdao Biotech Co., Ltd.).
sheet, expecting to provide a possible method for control of the molecule conformation and biodegradation improvement.
2.2. Recombinant GST-[GAGAGX]16 expression in E. coli BL21(DE3) Large-scale fermentations were performed with the E. coli BL21 expression strain. Competent cells of BL21 strain transformed with plasmids pGEX-KGA, pGEX-KGS, pGEX-KGY and pGEX-KGV were cultured on solid LB medium under ampicillin (100 µg/ml). A single colony was used to inoculate in 10 mL of sterile LB medium under ampicillin selection (100 µg/ml), and then amplified in fresh LBampicillin medium (1 L) until the OD600 reached approximately 0.9– 1.0 AU. Aqueous solution of IPTG was added to the cultures to a final concentration of 0.2 mM–1.0 mM to induce the synthesis of the target protein. The cells were harvested 5 h after induction by centrifugation at 4 °C, and four kinds of cell pellets were stored at −80 °C for further analysis. 2.3. Purification of recombinant GST-[GAGAGX]16 The ultimate recombinant GST-[GAGAGX]16 was purified through GST affinity Purification System(Novagen). The cell pellet was thawed on ice and resuspended in 100 ml of GST binding/washing buffer (4.3 mM Na2HPO4, 1.47 mM KH2PO4, 0.137 mM NaCl, 2.7 mM KCl, pH 7.3), then sonicated on ice with a Bioruptor sonicator (YJD-900, Shanghai Zhixing equipment Co., Ltd.) for lysis. Cell debris was removed by centrifugation at 4 °C. 25 ml of the supernatant was loaded onto a 4 ml GST affinity column, and then the column was washed with 50 ml GST washing buffer and placed on ice for 20 min. The fusion protein was eluted with 20 ml of GST elution buffer containing 10 mM reduced glutathione. 2.4. Cleavage of GST-[GAGAGX]16 with thrombin Evaporation and salting-out of the purified fusion proteins were performed on Centricon YM-3 spin filters (Amicon Bioseparations, Millipore) instrument with a molecular weight cutoff of 3000. Then
2.6. Preparation of sample for ThT fluorescence spectral analysis Amino acid analyses were carried out on a Hitachi L-800 Amino Acid Analyzer by hydrolyzing proteins in 6 M HCl at 150 °C for 85 min. All the liberated polypeptides were evaporated on Centricon YM-3 spin filters in PBS buffer (100 mM phosphate and 100 mM NaCl, pH 7.0) with 0.05% sodium azide and then filtered through 0.22 µm filters to remove any granular matter. The filtrates (approximately 3 mg/ml) were incubated in 1.5 ml sterile tubes for 0–8 days to assemble by themselves with continuous shaking at 37 °C. With the incubation progressing, aliquots (50 µl) of each uniform polypeptides solution were taken and stored at −20 °C at various time points. The time course of the aggregation process was monitored by a ThT (Ourchem, Shanghai Sinopharm Chemical Reagent Co., Ltd.) fluorescence assay on a fluorescence spectrometer (FLS920, EDINBURGH INSTRUMENTS). A ThT stock solution (100 µM) was prepared and filtered through a 0.22 µm filter. 20 µl of the incubated samples was added to 980 µl of 5 µl ThT in 50 mM glycine-NaOH buffer (pH 9.0). The emission intensities at 485 nm were recorded immediately after addition of the aliquots to the ThT solution with excitation at 446 nm. 3. Results and discussions 3.1. Construction for expression vector We utilized the commercially available expression vector pGEXKG, which places the synthetic genes under the control of the Ptac promoter and places a unique GST sequence at the N-terminus of the recombinant protein for purification by GST affinity chromatography. The complete extended genes were cloned into expression vector pGEX-KG-AgeI between HindIII/AgeI, resulting in plasmids (Fig. 2a, taking pGEX-KGS as an example) that contain the recombination genes expression cassette (Fig. 2b). A thrombin site was between GST and eGS (repeat). 3.2. Fusion proteins expression
Fig. 1. Designed DNA monomers.
pGEX-KGA, pGEX-KGS, pGEX-KGY and pGEX-KGV were transformed into E. coli BL21 and the encoded proteins were expressed upon induction by IPTG. Crude extracts were analyzed by polyacrylamide gel electrophoresis and Western blot analysis using an antiGST antibody. Staining of the gel with Coomassie Brilliant Blue
J.-N. Wang et al. / Materials Science and Engineering C 29 (2009) 1321–1325
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Fig. 2. Expression vector pGEX-KGS (a) and its expression cassette (b).
staining (Fig. 3a) and Western blot analysis (Fig. 3b) showed clear expression of fusion proteins. Expression vector is important for protein expression, especially hydrophobic and regular repeats. In our laboratory, we designed and constructed another expression vector pcDNA3.1B containing His-tag at N-terminal of polypeptides. All the fusion proteins could be expressed in E. coli and demonstrated by SDS–PAGE, Western blot analysis and ELISA [28], but expression levels were very low. Moreover, all products were synthesized as inclusion bodies, leading to the difficulty for expression, purification and following treatment: such as cleavage, purification, evaporation, salting-out. GST is an eximious hydrophilic protein, both the solubilities and expression levels of polypeptides combined GST-tag were obviously increased.
Fig. 3. SDS–PAGE (a) and Western blot analysis (b) of fusion proteins expressed in E. coli BL21. (a) The sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the fusion proteins by Coomassie blue staining. Lane 1, not containing the expression plasmid; Lane 2, containing the expression plasmid pGEX-KGV; Lane 3, containing the expression plasmid pGEX-KGY; Lane 4, containing the expression plasmid pGEX-KGS; Lane 5, containing the expression plasmid pGEX-KGA; Lane 6, protein molecular weight standards. (b) The Western blot results with anti-GST. Lane 1, containing the expression plasmid pGEX-KGA; Lane 2, containing the expression plasmid pGEX-KGS; Lane 3, containing the expression plasmid pGEX-KGY; Lane 4, containing the expression plasmid pGEX-KGV; Lane 5, not containing the expression plasmid.
3.3. Fusion proteins purification and liberation of polypeptides The fusion proteins were easily purified due to the GST-tag encoded by the pGEX-KG vector. The SDS–PAGE gel showed the purity of the fusion proteins following affinity chromatography (Fig. 4a), occasionally lower molecular weight bands were observed following purification by Western blot analysis (Data not shown). Thrombin is used to identify specifically amino acid sequence LVPRGS and cleave the amide linkage between Arg and Gly. The fusion proteins were subjected to thrombin cleavage in order to liberate the silk fibroin crystalline polypeptide analogues from the GST-tag. Since the N-terminal and C-terminal sequence comprised a small portion of the entire polypeptide chains of [GAGAGX]16 (less than 7% by residue), its presence should not significantly affect the overall conformational properties of the polypeptides. Thus, to a reasonable approximation, the silk fibroin crystalline repeat sequence or analogues should dominate the chemical and structural properties of cleaved polypeptides. Therefore,
Fig. 4. SDS–PAGE electrophoresis analysis of purified fusion proteins (a) and liberated polypeptides (b). (a) The sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of purified fusion proteins by Coomassie blue staining. Lane 1, protein molecular weight standards; Lane 2, fusion protein KGV; Lane 3, fusion protein KGY; Lane 4, fusion protein KGS; Lane 5, fusion protein KGA. (b) The sodium dodecyl sulfate/ polyacrylamide gel electrophoresis analysis of liberated polypeptides by Coomassie blue staining. Lane 1, liberated polypeptide eGV; Lane 2, liberated polypeptide eGY; Lane 3, liberated polypeptide eGS; Lane 4, liberated polypeptide eGA; Lane 5, protein molecular weight standards.
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Table 1 The amino acid compositions of the liberated polypeptides (mol%). Amino acid
eGA Theoretical
Experimental
eGS Theoretical
Experimental
eGY Theoretical
Experimental
Theoretical
Experimental
Gly Ala Ser Tyr Val Thr Gln Total
48.08 47.12 0.96 1.92 0.00 0.96 0.96 100.00
48.85 47.51 0.92 2.00 0.00 0.00 0.00 99.28
48.08 31.73 16.35 1.92 0.00 0.96 0.96 100.00
46.68 30.95 15.53 1.95 0.00 0.87 0.83 96.81
48.08 31.73 0.96 17.31 0.00 0.96 0.96 100.00
43.28 28.56 0.89 18.23 0.00 1.00 0.85 92.81
48.08 31.73 0.96 1.92 15.39 0.96 0.96 100.00
48.51 32.20 0.77 2.00 16.27 0.00 0.00 98.98
primary structural information gleaned from [GAGAGX]16 should provide insight into the secondary structure of the repetitive domains of silk-like fibroin crystals. Cleaved admixture containing GST and [GAGAGX]16 was loaded onto a GST affinity column once again binding GST. Evaporation and salting-out of the effluent were performed according to the method as described previously. Liberated polypeptides [GAGAGX]16 and their purity were assessed by SDS–PAGE electrophoresis (Fig. 4b). 3.4. Characterization of the liberated polypeptides The amino acid compositions of the liberated polypeptides [GAGAGX]16 were shown in Table 1. In all cases, the data were in very close accordance with the expected values and were taken as evidence that the correct polypeptides were expressed, purified and cleaved. Amino acid compositions reflected a high Gly + Ala content as
eGV
expected. The percentages of Gly and Ala differed by 3% or less from the predicted compositions except eGY, while the percentages of Ser, Tyr and Val from the eGS, eGY and eGV approximated to the calculated value. ThT fluorescence specifically and uniquely responds to the β-sheet structure, depending on the number of binding sites, affinity and quantum yield, so it is widely used for specific detection of stacked βsheet assemblies in the synthetical protein, and used to semiquantitatively estimate the relative rate of β-sheet formation [29,30]. Fig. 5 shows the fluorescence intensity in combination with ThT dyes at 485 nm of four polypeptides [GAGAGX]16 aggregations after incubation for 0-8 days. ThT fluorescence intensities from eGS and eGV were known to greatly increase fluorescence yields than that from eGA and eGY. Among the fluorescence spectral apices from the four polypeptides, eGS had the highest β-sheet content and eGV took the second place, while eGA had the lowest content, demonstrating that the polypeptides
Fig. 5. Time course of aggregation processes of four kinds of polypeptides as determined by the ThT fluorescence assay. 0→8: the liberated polypeptides from the GST fusion protiens were incubated fot 0-8 days with shaking at 37°C.
J.-N. Wang et al. / Materials Science and Engineering C 29 (2009) 1321–1325
repeated with [GAGAGS] and [GAGAGV] motifs were easier to form βsheet than repeated with [GAGAGA] and [GAGAGY] motifs. We further studied the stabilities of the four polypeptides by urea denaturation method, and the results showed that the most stable β-sheets were found in eGS and eGV (Data not shown). 4. Conclusions The four kinds of unique repetitive genes (less than 300 bp), derived from Fib-H gene or various analogs and composed of tandem repeats of one of the 18 bp oligonucleotides, were successfully cloned. This demonstrated the heritability and the stability of repetitive genes expression in E. coli. All the fusion proteins were expressed and purified as confirmed by SDS–PAGE electrophoresis and Western blot analysis. Among the four liberated polypeptides, eGS had the highest β-sheet content, and eGV took the second place, while eGA had the lowest content. This demonstrated that [GAGAGS] hexapeptide is the key basis to form β-sheet conformation in the silk fibroin crystalline domain. This study provided a possible method for control of the molecule conformation and biodegradation improvement of silk fibroin-based biomaterials by molecule design. This work was also aimed at studying the interaction between the amino acid compositions of silk-like repetitive crystalline protein and their structures or properties.
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Acknowledgments This work was supported by the grants from the National 973 Program of China (No. 2005CB623906), the Natural Science Foundation of Jiangsu province of China (No. BK2006054) and the Medicine Development Foundation of the Soochow University of China (No. EE 120613). We thank Prof. Changde Lu from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for kindly providing the research office and technical assistance and Prof. Junting Huang from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences for discussions and suggestions.
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Contents lists available at ScienceDirect
Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Biosynthesis and characterization of typical fibroin crystalline polypeptides of silkworm Bombyx mori ☆ Jian-Nan Wang a,⁎, Shu-Qin Yan a, Chang-De Lu b, Lun Bai a a b
College of Material Engineering, Soochow University, Suzhou 215021, PR China Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China
a r t i c l e
i n f o
Article history: Received 16 August 2007 Received in revised form 8 September 2008 Accepted 22 October 2008 Available online 5 November 2008 Keywords: Fibroin crystalline Silkworm Genes Fusion protein Thrombin ThT
a b s t r a c t We aimed to investigate the self-organization/self-assembly mechanisms of silkworm fibroin-based material. In the present study, for the first time, we designed and multimerized four DNA “monomer” sequences from structurally simple fibroin crystalline peptides or analog, [GAGAGX] (X = A, S, Y and V) to encode polypeptides [GAGAGX]16 (eGA, eGS, eGY and eGV) using a “head-to-tail” construction strategy. Multimers were cloned into pGEX-KG and fusion proteins GST-[GAGAGX]16 (KGA, KGS, KGY and KGV) were efficiently expressed in Escherichia coli. These fusion proteins were isolated and purified by GST affinity chromatography and confirmed by SDS–PAGE and Western blot analysis using antibody reactive to GST. The polypeptides were cleavaged from GST fusion proteins by digesting with thrombin enzyme. The composition of the four polypeptides was confirmed by composition analysis of amino acids, and their abilities to form βsheet structure were determined by ThT fluorescence spectral analysis. The content of β-sheet among the four polypeptides followed the order: eGS N eGV N eGY NeGA. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Silk fibroin of silkworm Bombyx mori is synthesized in the posterior silk gland cells of silkworm and secreted into the lumen. In recent years, silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility [1–5], slow degradability and remarkable mechanical properties of the material. An ideal medical material for tissue engineering should not only have good biological compatibility, but also possess appropriate porosity, and controlled structure and properties, especially controlled degradation [6]. Silk fibroin of B. mori is a crystal high polymer with three crystalline modifications, silkI, silkII and silkIII [7–13]. SilkII is the major formed part of stable β-sheet in natural silk fibroin [7,10] or regenerated silk fibroin [14,15], it is difficult for silk fibroin to be biodegraded [16,17]. Few studies focused on controlling the condensed structure of regenerated silk fibroin to improve degradation [18], but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes.
Abbreviations: Fib-H, silk fibroin heavy chain; Fib-L, silk fibroin light chain; ThT, thioflavin T; GST, glutathione transferase; G, Gly; A, Ala; S, Ser; Y, Tyr; V, Val; L, Leu; P, Pro; R, Arg. ☆ This work was completed at Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. ⁎ Corresponding author. Tel.: +86 512 67487067; fax: +86 512 67246786. E-mail address: [email protected] (J.-N. Wang). 0928-4931/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.10.029
Silk fibroin of the silkworm B. mori is a kind of structural protein. Protein architecture and mechanical properties are determined by its amino acid composition and sequence. Change in molecular composition by gene engineering would result in desired structural features and varied properties [19], including degradation. Each molecule of silk fibroin consists of Fib-H and Fib-L, which are linked by a disulfide bond of the two subunits [20], and another glycoprotein fibrohexamerin/P25 (FHX/P25), which associates with the H–L complex by hydrophobic interaction at a ratio of 1:6:6 to form an elementary unit of silk fibroin [21–24]. Fib-H is a hydrophobic protein composed of alternate arrays of 12 crystalline domains (repetitive domains) and 11 amorphous domains (non-repetitive domains), and its complete gene sequence and amino acid composition were recently determined by Zhou et al. [25,26]. The regular amino acid sequence of Fib-H, a crystalline domain, is distributed into several subdomains beginning with a stretch of [GAGAGS] hexapeptides and ending in [GAAS] tetrapeptide. [GAGAGY], [GAGAGA] and [GAGAGV] hexapeptides are also distributed in the crystalline domain, with the copy numbers of four hexapeptides 433, 120, 27 and 30. The [GAGAGS] hexapeptide assumes a β-sheet conformation and plays an important role in formation of better crystalline domains [27]. But functions of Tyr and Val or other amino acids in fibroin molecule conformation and property are still unclear. It is of great interest to study the modification of silk fibroin using genetic engineering. In the present study, we described the preparation of the polypeptides [GAGAGX]n (X = A, S, Y and V) containing highly repeated consensus 6 amino acid motif [GAGAGS] from silk fibroin crystalline of silkworm or various analog, and characterized their abilities to form β-
1322
J.-N. Wang et al. / Materials Science and Engineering C 29 (2009) 1321–1325
2. Experiments procedure
per milligram fusion protein was cleavaged with 1 U thrombin (Novagen) in 1× cleavage buffer at 20 °C for 16 h. The reaction mixture was diluted in GST binding buffer and loaded onto GST affinity column to remove GST-tag.
2.1. Synthetic gene assembly expression vector constructs
2.5. SDS–PAGE electrophoresis and Western blot analysis
The DNA monomers of 18 base pairs (Fig. 1, underlined) derived from Fib-H genes and encoding hexapeptides GAGAGX(X = A, S, Y and V) were designed and then polymerized to 16 repeats by repeated doubling in Escherichia coli (E. coli). Multimers were formed by insertion of ScaI/NgoMIV digested fragments into ScaI/AgeI digested vectors, these enzymes generate identical cohesive ends, and when the ligation joins a NgoMIV and a AgeI site together in a “head-to-tail” fashion, any internal restriction sites are destroyed. The extended genes, flanked by AgeI and HindIII sites, were inserted into the expression vector pGEX-KG-AgeI at the same sites following the gene sequence encoding GST. pGEX-KG-AgeI was derived from pGEX-KG by inserting synthetic double-stranded oligonucleotides (5’ GATCCACCGGTGG 3’, 5’ AATTCCACCGGTG 3’) fragment containing a AgeI site (underlined). The resulting plasmids were designated pGEX-KGA, pGEX-KGS, pGEX-KGY and pGEX-KGV. All cloned fragments were further verified by nucleotide sequencing (Invitrogen).
The protein content within the whole cell lysate, the purified fusion protein and the liberated polypeptide were identified on SDS– PAGE of 12% polyacrylamide gel (Sigma) by adding equal volume of sample buffer (20% 2-mercaptoethanol; 4% SDS; 20% glycerol; 0.2% bromophenol blue; 0.1 M Tris–HCl, pH 6.8) and boiling for 3–5 min, finally stained by Coomassie brilliant blue. After SDS–PAGE, the proteins (within the whole cell lysate) were transferred onto a PVDF membrane(Immobilon-P, Millipore), and subsequently blocked with TBST (50 mM Tris–HCl, pH 7.6; 150 mM NaCl; 0.05% Tween-20) containing 5% of nonfat dried milk. Then membrane was incubated in TBST containing 5% of nonfat dried milk and 1000× diluted GST antibody (Stressgen) for 1 h, after washing with TBST three times (each for 10 min), followed incubation of horseradish peroxidase labeled rabbit IgG antibody (Jingmei Biotech Co., Ltd.). The antibody was detected using DAB reagent (Shanghai Changdao Biotech Co., Ltd.).
sheet, expecting to provide a possible method for control of the molecule conformation and biodegradation improvement.
2.2. Recombinant GST-[GAGAGX]16 expression in E. coli BL21(DE3) Large-scale fermentations were performed with the E. coli BL21 expression strain. Competent cells of BL21 strain transformed with plasmids pGEX-KGA, pGEX-KGS, pGEX-KGY and pGEX-KGV were cultured on solid LB medium under ampicillin (100 µg/ml). A single colony was used to inoculate in 10 mL of sterile LB medium under ampicillin selection (100 µg/ml), and then amplified in fresh LBampicillin medium (1 L) until the OD600 reached approximately 0.9– 1.0 AU. Aqueous solution of IPTG was added to the cultures to a final concentration of 0.2 mM–1.0 mM to induce the synthesis of the target protein. The cells were harvested 5 h after induction by centrifugation at 4 °C, and four kinds of cell pellets were stored at −80 °C for further analysis. 2.3. Purification of recombinant GST-[GAGAGX]16 The ultimate recombinant GST-[GAGAGX]16 was purified through GST affinity Purification System(Novagen). The cell pellet was thawed on ice and resuspended in 100 ml of GST binding/washing buffer (4.3 mM Na2HPO4, 1.47 mM KH2PO4, 0.137 mM NaCl, 2.7 mM KCl, pH 7.3), then sonicated on ice with a Bioruptor sonicator (YJD-900, Shanghai Zhixing equipment Co., Ltd.) for lysis. Cell debris was removed by centrifugation at 4 °C. 25 ml of the supernatant was loaded onto a 4 ml GST affinity column, and then the column was washed with 50 ml GST washing buffer and placed on ice for 20 min. The fusion protein was eluted with 20 ml of GST elution buffer containing 10 mM reduced glutathione. 2.4. Cleavage of GST-[GAGAGX]16 with thrombin Evaporation and salting-out of the purified fusion proteins were performed on Centricon YM-3 spin filters (Amicon Bioseparations, Millipore) instrument with a molecular weight cutoff of 3000. Then
2.6. Preparation of sample for ThT fluorescence spectral analysis Amino acid analyses were carried out on a Hitachi L-800 Amino Acid Analyzer by hydrolyzing proteins in 6 M HCl at 150 °C for 85 min. All the liberated polypeptides were evaporated on Centricon YM-3 spin filters in PBS buffer (100 mM phosphate and 100 mM NaCl, pH 7.0) with 0.05% sodium azide and then filtered through 0.22 µm filters to remove any granular matter. The filtrates (approximately 3 mg/ml) were incubated in 1.5 ml sterile tubes for 0–8 days to assemble by themselves with continuous shaking at 37 °C. With the incubation progressing, aliquots (50 µl) of each uniform polypeptides solution were taken and stored at −20 °C at various time points. The time course of the aggregation process was monitored by a ThT (Ourchem, Shanghai Sinopharm Chemical Reagent Co., Ltd.) fluorescence assay on a fluorescence spectrometer (FLS920, EDINBURGH INSTRUMENTS). A ThT stock solution (100 µM) was prepared and filtered through a 0.22 µm filter. 20 µl of the incubated samples was added to 980 µl of 5 µl ThT in 50 mM glycine-NaOH buffer (pH 9.0). The emission intensities at 485 nm were recorded immediately after addition of the aliquots to the ThT solution with excitation at 446 nm. 3. Results and discussions 3.1. Construction for expression vector We utilized the commercially available expression vector pGEXKG, which places the synthetic genes under the control of the Ptac promoter and places a unique GST sequence at the N-terminus of the recombinant protein for purification by GST affinity chromatography. The complete extended genes were cloned into expression vector pGEX-KG-AgeI between HindIII/AgeI, resulting in plasmids (Fig. 2a, taking pGEX-KGS as an example) that contain the recombination genes expression cassette (Fig. 2b). A thrombin site was between GST and eGS (repeat). 3.2. Fusion proteins expression
Fig. 1. Designed DNA monomers.
pGEX-KGA, pGEX-KGS, pGEX-KGY and pGEX-KGV were transformed into E. coli BL21 and the encoded proteins were expressed upon induction by IPTG. Crude extracts were analyzed by polyacrylamide gel electrophoresis and Western blot analysis using an antiGST antibody. Staining of the gel with Coomassie Brilliant Blue
J.-N. Wang et al. / Materials Science and Engineering C 29 (2009) 1321–1325
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Fig. 2. Expression vector pGEX-KGS (a) and its expression cassette (b).
staining (Fig. 3a) and Western blot analysis (Fig. 3b) showed clear expression of fusion proteins. Expression vector is important for protein expression, especially hydrophobic and regular repeats. In our laboratory, we designed and constructed another expression vector pcDNA3.1B containing His-tag at N-terminal of polypeptides. All the fusion proteins could be expressed in E. coli and demonstrated by SDS–PAGE, Western blot analysis and ELISA [28], but expression levels were very low. Moreover, all products were synthesized as inclusion bodies, leading to the difficulty for expression, purification and following treatment: such as cleavage, purification, evaporation, salting-out. GST is an eximious hydrophilic protein, both the solubilities and expression levels of polypeptides combined GST-tag were obviously increased.
Fig. 3. SDS–PAGE (a) and Western blot analysis (b) of fusion proteins expressed in E. coli BL21. (a) The sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the fusion proteins by Coomassie blue staining. Lane 1, not containing the expression plasmid; Lane 2, containing the expression plasmid pGEX-KGV; Lane 3, containing the expression plasmid pGEX-KGY; Lane 4, containing the expression plasmid pGEX-KGS; Lane 5, containing the expression plasmid pGEX-KGA; Lane 6, protein molecular weight standards. (b) The Western blot results with anti-GST. Lane 1, containing the expression plasmid pGEX-KGA; Lane 2, containing the expression plasmid pGEX-KGS; Lane 3, containing the expression plasmid pGEX-KGY; Lane 4, containing the expression plasmid pGEX-KGV; Lane 5, not containing the expression plasmid.
3.3. Fusion proteins purification and liberation of polypeptides The fusion proteins were easily purified due to the GST-tag encoded by the pGEX-KG vector. The SDS–PAGE gel showed the purity of the fusion proteins following affinity chromatography (Fig. 4a), occasionally lower molecular weight bands were observed following purification by Western blot analysis (Data not shown). Thrombin is used to identify specifically amino acid sequence LVPRGS and cleave the amide linkage between Arg and Gly. The fusion proteins were subjected to thrombin cleavage in order to liberate the silk fibroin crystalline polypeptide analogues from the GST-tag. Since the N-terminal and C-terminal sequence comprised a small portion of the entire polypeptide chains of [GAGAGX]16 (less than 7% by residue), its presence should not significantly affect the overall conformational properties of the polypeptides. Thus, to a reasonable approximation, the silk fibroin crystalline repeat sequence or analogues should dominate the chemical and structural properties of cleaved polypeptides. Therefore,
Fig. 4. SDS–PAGE electrophoresis analysis of purified fusion proteins (a) and liberated polypeptides (b). (a) The sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of purified fusion proteins by Coomassie blue staining. Lane 1, protein molecular weight standards; Lane 2, fusion protein KGV; Lane 3, fusion protein KGY; Lane 4, fusion protein KGS; Lane 5, fusion protein KGA. (b) The sodium dodecyl sulfate/ polyacrylamide gel electrophoresis analysis of liberated polypeptides by Coomassie blue staining. Lane 1, liberated polypeptide eGV; Lane 2, liberated polypeptide eGY; Lane 3, liberated polypeptide eGS; Lane 4, liberated polypeptide eGA; Lane 5, protein molecular weight standards.
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Table 1 The amino acid compositions of the liberated polypeptides (mol%). Amino acid
eGA Theoretical
Experimental
eGS Theoretical
Experimental
eGY Theoretical
Experimental
Theoretical
Experimental
Gly Ala Ser Tyr Val Thr Gln Total
48.08 47.12 0.96 1.92 0.00 0.96 0.96 100.00
48.85 47.51 0.92 2.00 0.00 0.00 0.00 99.28
48.08 31.73 16.35 1.92 0.00 0.96 0.96 100.00
46.68 30.95 15.53 1.95 0.00 0.87 0.83 96.81
48.08 31.73 0.96 17.31 0.00 0.96 0.96 100.00
43.28 28.56 0.89 18.23 0.00 1.00 0.85 92.81
48.08 31.73 0.96 1.92 15.39 0.96 0.96 100.00
48.51 32.20 0.77 2.00 16.27 0.00 0.00 98.98
primary structural information gleaned from [GAGAGX]16 should provide insight into the secondary structure of the repetitive domains of silk-like fibroin crystals. Cleaved admixture containing GST and [GAGAGX]16 was loaded onto a GST affinity column once again binding GST. Evaporation and salting-out of the effluent were performed according to the method as described previously. Liberated polypeptides [GAGAGX]16 and their purity were assessed by SDS–PAGE electrophoresis (Fig. 4b). 3.4. Characterization of the liberated polypeptides The amino acid compositions of the liberated polypeptides [GAGAGX]16 were shown in Table 1. In all cases, the data were in very close accordance with the expected values and were taken as evidence that the correct polypeptides were expressed, purified and cleaved. Amino acid compositions reflected a high Gly + Ala content as
eGV
expected. The percentages of Gly and Ala differed by 3% or less from the predicted compositions except eGY, while the percentages of Ser, Tyr and Val from the eGS, eGY and eGV approximated to the calculated value. ThT fluorescence specifically and uniquely responds to the β-sheet structure, depending on the number of binding sites, affinity and quantum yield, so it is widely used for specific detection of stacked βsheet assemblies in the synthetical protein, and used to semiquantitatively estimate the relative rate of β-sheet formation [29,30]. Fig. 5 shows the fluorescence intensity in combination with ThT dyes at 485 nm of four polypeptides [GAGAGX]16 aggregations after incubation for 0-8 days. ThT fluorescence intensities from eGS and eGV were known to greatly increase fluorescence yields than that from eGA and eGY. Among the fluorescence spectral apices from the four polypeptides, eGS had the highest β-sheet content and eGV took the second place, while eGA had the lowest content, demonstrating that the polypeptides
Fig. 5. Time course of aggregation processes of four kinds of polypeptides as determined by the ThT fluorescence assay. 0→8: the liberated polypeptides from the GST fusion protiens were incubated fot 0-8 days with shaking at 37°C.
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repeated with [GAGAGS] and [GAGAGV] motifs were easier to form βsheet than repeated with [GAGAGA] and [GAGAGY] motifs. We further studied the stabilities of the four polypeptides by urea denaturation method, and the results showed that the most stable β-sheets were found in eGS and eGV (Data not shown). 4. Conclusions The four kinds of unique repetitive genes (less than 300 bp), derived from Fib-H gene or various analogs and composed of tandem repeats of one of the 18 bp oligonucleotides, were successfully cloned. This demonstrated the heritability and the stability of repetitive genes expression in E. coli. All the fusion proteins were expressed and purified as confirmed by SDS–PAGE electrophoresis and Western blot analysis. Among the four liberated polypeptides, eGS had the highest β-sheet content, and eGV took the second place, while eGA had the lowest content. This demonstrated that [GAGAGS] hexapeptide is the key basis to form β-sheet conformation in the silk fibroin crystalline domain. This study provided a possible method for control of the molecule conformation and biodegradation improvement of silk fibroin-based biomaterials by molecule design. This work was also aimed at studying the interaction between the amino acid compositions of silk-like repetitive crystalline protein and their structures or properties.
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Acknowledgments This work was supported by the grants from the National 973 Program of China (No. 2005CB623906), the Natural Science Foundation of Jiangsu province of China (No. BK2006054) and the Medicine Development Foundation of the Soochow University of China (No. EE 120613). We thank Prof. Changde Lu from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences for kindly providing the research office and technical assistance and Prof. Junting Huang from the Sericultural Research Institute, Chinese Academy of Agricultural Sciences for discussions and suggestions.
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