Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the C-terminal.Its effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a reporter.Secretion of EGFP fusion proteinswas examined under fluorescence microscope.FibH SPs with lengths of 20,18,16 and 12 a.a.can directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP.When 16a.a.of the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk gland.The cleavage site of the SP is discussed andcompared with the predictive results of the SignalP 3.0 online prediction program.     

Recognition of signal peptide by protein translocation machinery in middle silk gland of silkworm Bombyx mori

To investigate the functions of signal peptide in protein secretion in the middle silk gland of silkworm Bombyx mori, a series of recombinant Autographa californica multiple nucleopolyhedroviruses containing enhanced green fluorescent protein （egfp） gene, led by sericin-1 promoter and mutated signal peptide coding sequences, were constructed by region-deletions or single amino acid residue deletions. The recombinant Autographa californica multiple nucleopolyhedroviruses were injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of EGFP in the middle silk gland were examined by fluorescence microscopy and Western blot analysis. Results showed that even with a large part （up to 14 amino acid residues） of the ser-1 signal peptide deleted, the expressed EGFP could still be secreted into the cavity of the silk gland. Western blot analysis showed that shortening of the signal peptide from the C-terminal suppressed the maturation of pro-EGFP to EGFP. When 8 amino acid residues were deleted from the C-terminal of the signal peptide （mutant 13 aa）, the secretion of EGFP was incomplete, implicating the importance of proper coupling of the h-region and c-region. The deletion of amino acid residue（s） in the h-region did not affect the secretion of EGFP, indicating that the recognition of signal peptide by translocation machinery was mainly by a structural domain, but not by special amino acid residue（s）. Furthermore, the deletion ofArg^2 or replacement with Asp in the n-region of the signal peptide did not influence secretion of EGFP, suggesting that a positive charge is not crucial.     

A new method for the modification of fibroin heavy chain protein in the transgenic silkworm

We constructed a new plasmid vector for the production of a modified silk fibroin heavy chain protein (H-chain) in the transgenic silkworm. The plasmid (pHC-null) contained the promoter and the 3' region of a gene encoding the H-chain and the coding regions for the N-terminal domain and the C-terminal domain of the H-chain. For the model protein, we cloned a foreign gene that encoded EGFP between the N-terminal domain and the C-terminal domain in pHC-null and generated transgenic silkworms that produced a modified H-chain, HC-EGFP. Transgenic silkworms produced HC-EGFP in the posterior part of silk gland cells, secreted it into the lumen of the gland, and produced a cocoon with HC-EGFP as part of the fibroin proteins. N-terminal sequencing of HC-EGFP localized the signal sequence cleavage site to between positions A((21)) and N((22)). These results indicate that our new plasmid successfully produced the modified H-chain in a transgenic silkworm.     

Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation

The determinants governing the self-catalyzed splicing and cleavage events by a mini-intein of 154 amino acids, derived from the dnaB gene of Synechocystis sp. were investigated. The residues at the splice junctions have a profound effect on splicing and peptide bond cleavage at either the N- or C-terminus of the intein. Mutation of the native Gly residue preceding the intein blocked splicing and cleavage at the N-terminal splice junction, while substitution of the intein C-terminal Asn154 resulted in the modulation of N-terminal cleavage activity. Controlled cleavage at the C-terminal splice junction involving cyclization of Asn154 was achieved by substitution of the intein N-terminal cysteine residue with alanine and mutation of the native C-extein residues. The C-terminal cleavage reaction was found to be pH-dependent, with an optimum between pH6.0 and 7.5. These findings allowed the development of single junction cleavage vectors for the facile production of proteins as well as protein building blocks with complementary reactive groups. A protein sequence was fused to either the N-terminus or C-terminus of the intein, which was fused to a chitin binding domain. The N-terminal cleavage reaction was induced by 2-mercaptoethanesulfonic acid and released the 43kDa maltose binding protein with an active C-terminal thioester. The 58kDa T4 DNA ligase possessing an N-terminal cysteine was generated by a C-terminal cleavage reaction induced by pH and temperature shifts. The intein-generated proteins were joined together through a native peptide bond. This intein-mediated protein ligation approach opens up novel routes in protein engineering.     

The translocation inhibitor CAM741 interferes with vascular cell adhesion molecule 1 signal peptide insertion at the translocon

The cyclopeptolide CAM741 selectively inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), a process that is dependent on its signal peptide. In this study we identified the C-terminal (C-) region upstream of the cleavage site of the VCAM1 signal peptide as most critical for inhibition of translocation by CAM741, but full sensitivity to the compound also requires residues of the hydrophobic (h-) region and the first amino acid of the VCAM1 mature domain. The murine VCAM1 signal peptide, which is less susceptible to translocation inhibition by CAM741, can be converted into a fully sensitive signal peptide by two amino acid substitutions identified as critical for compound sensitivity of the human VCAM1 signal peptide. Using cysteine substitutions of non-critical residues in the human VCAM1 signal peptide and chemical cross-linking of targeted short nascent chains we show that, in the presence of CAM741, the N- and C-terminal segments of the VCAM1 signal peptide could be cross-linked to the cytoplasmic tail of Sec61beta, indicating altered positioning of the VCAM1 signal peptide relative to this translocon component. Moreover, translocation of a tag fused N-terminal to the VCAM1 signal peptide is selectively inhibited by CAM741. Our data indicate that the compound inhibits translocation of VCAM1 by interfering with correct insertion of its signal peptide into the translocon.     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin

The secretion signal of extracellular metalloprotease B that is secreted without a signal peptide by the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi is shown by deletion and gene fusion analyses to be located within the last 40 C-terminal amino acids. Secretion of a peptide containing only this region of the protease requires the same three secretion factors (PrtD, PrtE, and PrtF) that were previously shown to be required for the secretion of the full-length protease. This secretion signal can also be recognized, albeit inefficiently, by the analogous secretion machinery of alpha-hemolysin, another protein with a C-terminal secretion signal that is secreted by some strains of the Gram-negative bacterium Escherichia coli. The secretion signal was fused to an internal 200-amino acid fragment from the sequence of the cytoplasmic protein amylomaltase to promote its specific secretion by the protease secretion pathway. Almost exactly the same sequence as that identified as the protease B secretion signal was also found at the C terminus of metalloprotease C that is also secreted by E. chrysanthemi.     

Identification of a functionally relevant signal peptide of mouse ficolin A

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.     

Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield

Spider dragline silk is a unique fibrous protein with combination of tensile strength and elasticity, but the isolation of large amount of silk from spiders is not feasible. In this paper, we used a newly established Bac-to-Bac/BmNPV Baculovirus expression system to express the recombinant spider (Nephila clavata) dragline silk protein (MaSp1) fused EGFP in BmN cells and larvae of silkworm. A 70 kDa fusion protein was visualized after rBacmid/BmNPV/drag infection by SDS-PAGE and immunoblotting analysis. Fusion protein expressed in the BmN cells probably occupied five percent of the cell total protein; In a silkworm larva, approximately 6 mg fusion proteins were expressed. Solubility analysis of the expressed spider dragline silk protein indicated that 60% fusion protein is insoluble. EGFP fluorescence showed that fusion protein is tend to form aggregate by self assemblage. The results indicated the solubility is the primary limit for spider dragline proteins yield. It also suggested that directly produce fibrous spider silk in the secreting-silk organs of the transgenic silkworm larvae might be a better method.     

Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system

We constructed the fibroin H-chain expression system to produce recombinant proteins in the cocoon of transgenic silkworms. Feline interferon (FeIFN) was used for production and to assess the quality of the product. Two types of FeIFN fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the FeIFN/H-chain fusion gene was regulated by the fibroin H-chain promoter domain. The transgenic silkworms introduced these constructs with the piggyBac transposon-derived vector, which produced the normal sized cocoons containing each FeIFN/H-chain fusion protein. Although the native-protein produced by transgenic silkworms have almost no antiviral activity, the proteins after the treatment with PreScission protease to eliminate fibroin H-chain derived N- and C-terminal sequences from the products, had very high antiviral activity. This H-chain expression system, using transgenic silkworms, could be an alternative method to produce an active recombinant protein and silk-based biomaterials.     

Relation of the Escherichia coli dnaX gene to its two products--the tau and gamma subunits of DNA polymerase III holoenzyme.

The Escherichia coli DNA polymerase III holoenzyme 71.1 kDa tau subunit is a 643 amino acid protein encoded by the dnaX gene. This gene also encodes the holoenzyme 56.5 kDa gamma subunit. The tau factor (as a tau'-LacZ' fusion protein) has been isolated and shown to be cleaved in vitro to form gamma and a 135 kda C-terminal cleavage product. The tau'-LacZ' fusion protein, gamma, and the C-terminal cleavage product have been isolated. N-terminal sequencing has demonstrated that tau and gamma share the same N-terminal sequences and that tau is proteolytically cleaved in vitro between residues 498 and 499 to form gamma. In addition, residues 420-440 were shown to be present in both tau and gamma by use of antibody specific for a synthetic peptide corresponding to that sequence. Some mechanism functions in vivo to ensure that tau and gamma are synthesized in a ratio of about one-to-one, as shown by radioimmune precipitation of tau and gamma from cellular extracts.     

Escherichia coli expression and processing of human interleukin-1 beta fused to signal peptides

Escherichia coli expression, processing, and secretion of human interleukin-1 beta (IL-1 beta) fused to the signal peptide of E. coli OmpA or PhoA protein were studied. With fusion to either signal sequence, high-level expression was observed and the products accumulated to about 20% of total cell protein. In the fusion to OmpA leader sequence, more than 50% of the product has the OmpA signal peptide removed precisely. The majority of the processed material is not released by osmotic shock. On the other hand, very little of the material from the fusion to PhoA has the PhoA signal peptide removed. Use of the host with a mutation in prlA or prlF, variation of temperature for cell growth, and alteration of the amino acid residues around the cleavage site do not facilitate processing of the PhoA signal peptide. These results suggest that some component in the PhoA signal peptide, interacting with the Il-1 beta sequence, is interfering with the processing of the signal peptide.     

Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.     

Secretion of thermophilic bacterial cellobiohydrolase in Saccharomyces cerevisiae

The partial DNA sequence corresponding to the N-terminal amino acid sequence of cellobiohydrolase derived from a thermophilic anaerobe NA10 was determined. The cellobiohydrolase gene fused to the secretion signal (signal peptide and T-S region) from Saccharomyces diastaticus was expressed in an ethanologenic yeast, S. cerevisiae YIY345, under control of the glucoamylase promoter. The recombinant yeast produced cellobiohydrolase: approximately 40% of the total cellobiohydrolase activity was detected in the medium, and the remaining cellobiohydrolase was localized in the intracellular fraction. An analysis of the N-terminal amino acid sequence of the main intracellular cellobiohydrolase revealed that the signal peptide and T-S region were removed proteolytically. Alteration of the amino acid residues at the cleavage site by insertion of a Bgl II linker led to an approximately 3.5-fold increase in the total cellobiohydrolase production, but did not affect the efficiency of secretion into the medium. Cellobiohydrolase production was not repressed in the presence of glucose. The recombinant yeast hydrolyzed carboxymethyl cellulose in the medium. The results suggest the possibility of the direct bioconversion of cellulose to ethanol by the recombinant yeast.     

Signal peptide of Lassa virus glycoprotein GP-C exhibits an unusual length

Lassa virus glycoprotein is synthesized as precursor GP-C into the lumen of the endoplasmic reticulum and cleaved posttranslationally into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by subtilase SKI-1/S1P. The N-terminal portion of the primary translation product preGP-C contains a signal peptide of unknown length. In order to demonstrate the signal peptide cleavage site, purified viral GP-1 isolated from Lassa virus particles was N-terminally sequenced as TSLYKGV, identical to amino acids 59-65 of GP-C. Mutational analysis of the amino acid residues flanking the putative cleavage site led to non-cleavable preGP-C indicating that no other signal peptide cleavage site exists. Interestingly, GP-C mutants with a non-cleavable signal peptide were not further processed by SKI-1/S1P. This observation suggests that the signal peptide cleavage is necessary for GP-C maturation and hence for Lassa virus replication.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).