Conformation transition kinetics of Bombyx mori silk protein

Time-resolved FTIR analysis was used to monitor the conformation transition induced by treating regenerated Bombyx mori silk fibroin films and solutions with different concentrations of ethanol. The resulting curves showing the kinetics of the transition for both films and fibroin solutions were influenced by the ethanol concentration. In addition, for silk fibroin solutions the protein concentration also had an effect on the kinetics. At low ethanol concentrations (for example, less than 40% v/v in the case of film), films and fibroin solutions showed a phase in which beta-sheets slowly formed at a rate dependent on the ethanol concentration. Reducing the concentration of the fibroin in solutions also slowed the formation of beta-sheets. These observations suggest that this phase represents a nucleation step. Such a nucleation phase was not seen in the conformation transition at ethanol concentrations > 40% in films or > 50% in silk fibroin solutions. Our results indicate that the ethanol-induced conformation transition of silk fibroin in films and solutions is a three-phase process. The first phase is the initiation of beta-sheet structure (nucleation), the second is a fast phase of beta-sheet growth while the third phase represents a slow perfection of previously formed beta-sheet structure. The nucleation step can be very fast or relatively slow, depending on factors that influence protein chain mobility and intermolecular hydrogen bond formation. The findings give support to the previous evidence that natural silk spinning in silkworms is nucleation-dependent, and that silkworms (like spiders) use concentrated silk protein solutions, and careful control of the pH value and metallic ion content of the processing environment to speed up the nucleation step to produce a rapid conformation transition to convert the water soluble spinning dope to a tough solid silk fiber.     

Nucleic acids accumulation of silk gland of Bombyx mori in relation to silk protein

• 1.1. Productivity of silk, properly fibroin, of the silkworm Bombyx mori was in proportion to the amount of RNA accumulation in the posterior division of silk gland.
• 2.2. DNA content of the silk gland of a line of high silk productivity was twice as much as that of low productivity. A DNA molecule can transcribe RNA, ranged from 3 × 10⁶ to 6 × 10⁶ molecules.
• 3.3. An application of actinomycin to larvae lowered an accumulation of RNA in the silk gland and resulted in a decrease of silk production.
• 4.4. Even under the upper limit of starvation by which the larvae complete their life, the silk gland kept a normal level of the DNA content, except that it lost the synthesizing activity of RNA.
• 5.5. Treatment of larvae with juvenile hormone occasionally induced another DNA replication of the silk gland.

     

MicroRNA expression profiling of the fifth-instar posterior silk gland of Bombyx mori

Background

The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production.

Results

Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, of the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the targets into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing.

Conclusion

The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs and their targets in silk production.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-410) contains supplementary material, which is available to authorized users.     

Structure refinement and diffuse streak scattering of silk (Bombyx mori).

Reexamination of the crystal structure of silk (Bombyx mori) was carried out by X-ray diffraction method. Four molecular chains are contained in the rectangular unit cell with parameters, a = 9.38 A, b = 9.49 A, and c (fiber axis) = 6.98 A, and the space group P2(1)-C(2)2. Silk assumes the statistical crystal structure, in which two antipolar-antiparallel sheet structures with different orientations statistically occupy a crystal site with the ratio 2:1. The molecular conformation is essentially the same pleated sheet structure as Marsh, Corey and Pauling proposed. However, the sheet structure formed by hydrogen bonds assumes the antipolar antiparallel structure different from that proposed by Marsh, Corey and Pauling, in which the methyl groups of alanine residues alternately point to both sides of the sheet structure along the hydrogen bonding direction. The crystalline region of silk is composed of stacking of two antipolar antiparallel sheet structures with different orientations.     

纳米纤维家蚕丝蛋白软支架制备及性能

【目的】通过调控蚕丝蛋白溶液的状态,可实现对蚕丝蛋白支架的力学及结构性能的调控,获得适宜于内皮细胞生长的支架材料,解决复杂组织修复中由于不利于组织长入和血管生长而导致组织功能无法修复的问题。【方法】向新鲜蚕丝蛋白溶液中添加蚕丝蛋白纳米纤维增加冻干支架的成孔性,基于丝蛋白自组装原理对丝素蛋白溶液进行浓缩处理增加分子间亲水作用力,获得不同状态的丝蛋白溶液,最后冷冻干燥,制备支架,观察蚕丝蛋白状态变化对支架性能的影响。【结果】实验结果表明,蚕丝蛋白溶液经浓缩处理可以降低支架Silk II的形成,获得较柔软支架。【结论】添加蚕丝蛋白纳米纤维并浓缩处理可以获得模量适宜内皮细胞生长的支架材料,为不同软组织的修复提供了良好的基质材料。     

Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori

Three alleles of the sericin (Ser) 2-encoding gene (Ser2), called L, C and mC, were isolated from a Bombyx mori genomic library, and two related ones, called mCL and Cv, were also characterized in B. mori European strains. The Ser2 gene gives rise to two middle silk gland mRNAs by differential splicing. The size of a short mRNA (3.1 kb) is constant, but the length of a longer one ranges from 5 to 6.4 kb depending on the Ser2 allele. These length variations probably result from unequal recombinations in a region which contains about 30 well conserved 45-bp repeats coding for a Ser-like peptide. Furthermore, the L allele (and probably the mCL one) contains a 4.4-kb retrotransposon, resembling the copia-like ones of Drosophila.     

Structure of Bombyx mori chemosensory protein 1 in solution

Chemosensory Proteins (CSPs) represent a family of conserved proteins found in insects that may be involved in chemosensory functions. BmorCSP1 is expressed mainly in antennae and legs of the silkworm moth Bombyx mori and was cloned from antennal cDNA. Here we report the determination of the structure of Bombyx mori CSP1 (BmorCSP1) by NMR. The overall fold of BmorCSP1 is globular and comprises six alpha-helices. These helices span residues 10-14, 17-27, 35-49, 57-72, 75-85, and 92-100. The internal hydrophobic sides of the helices are formed mostly by leucine and isoleucine residues and, therefore, well suited to constitute a binding site for hydrophobic ligands.     

Materials fabrication from Bombyx mori silk fibroin

Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.     

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori

Small non-protein coding RNAs （ncRNAs） play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs （four small nucleolar RNAs and five unclassified ncRNAs） that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm- 152, exhibited converse expression pattem with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.     

The genes for silk fibroin in Bombyx mori

The genes for the protein silk fibroin were quantitated by hybridizaton of purified fibroin messenger RNA with the DNA from several tissues of the silk-worm Bombyx mori.     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

Copper in the silk formation process of Bombyx mori silkworm

Evidence is presented here that cupric ions play a part in the natural spinning of Bombyx mori silk. Proton induced X-ray emission studies revealed that the copper content increased from the posterior part to the anterior part of silk gland, and then further increased in the silk fiber. Spectrophotometric analysis demonstrated that cupric ions formed coordination complexes with silk fibroin chains while Raman spectroscopy indicated that they induced a conformation transition from random coil/helix to beta-sheet. Taken together these findings indicate that copper could play a role in the natural spinning process in silkworms.     

家蚕前部丝腺特异表皮蛋白Bm11721的鉴定及表达

家蚕的丝腺是其丝蛋白合成和分泌的器官,根据其形态和功能的不同分为前部、中部和后部丝腺,前部丝腺不具有合成丝蛋白的能力,是丝蛋白构象发生转变的场所。剪切力在丝蛋白构象转变中起到重要的作用,其在家蚕前部丝腺主要由前部丝腺逐渐变细的管腔结构和富含几丁质及表皮蛋白的坚硬的内壁提供。鉴定家蚕前部丝腺新的几丁质结合蛋白,并调查其在家蚕幼虫不同组织的表达特征。通过几丁质亲和层析的方法在前部丝腺筛选并鉴定到一个新的具有几丁质结合功能的表皮蛋白Bm11721,其编码基因编号为BGIBMGA011721(Gen Bank Accession No.NM-001173285.1)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm11721的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm11721均只在前部丝腺特异表达,且Bm11721蛋白在5龄期的前部丝腺中恒定表达。免疫荧光定位结果显示Bm11721蛋白定位在前部丝腺的内膜中,推测其可能与前部丝腺的机械硬度有关,为丝蛋白的构象转变提供剪切力。     

The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori

The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It’s a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.     

Structure and expression of mRNA for a pupal cuticle protein of the silkworm,Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.