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1.
Aqualysin I is a subtilisin-type serine protease secreted into the medium by Thermus aquaticus YT-1. Thermus thermophilus cells harboring a plasmid for the aqualysin I precursor secreted pro-aqualysin I with the C-terminal pro-sequence into the culture medium, and the precursor was then processed to the mature enzyme during the cultivation. However, the extracellular levels of aqualysin I in T. thermophilus cells harboring plasmids for deletion mutants as to the C-terminal pro-sequence were about 10–20% in comparison with the level of wild-type. Only the mature enzyme could be detected in the medium, while pro-aqualysin I with the C-terminal pro-sequence could not. These results suggest that the C-terminal pro-sequence of aqualysin I plays an important role in the extracellular secretion of aqualysin I.  相似文献   

2.
The precursor of aqualysin I, an extracellular subtilisin-type protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, a protease domain, and a C-terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C-terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS-polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C-terminal extended sequence occurs through an intermolecular self-processing mechanism.  相似文献   

3.
Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.  相似文献   

4.
 The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F′ episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl β-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I productivity for a fed-batch culture using two carbon sources, glucose and glycerol, reached more than 150 kU/ml enzymatically active aqualysin I. Received: 19 May 1995/Received revision: 28 July 1995/Accepted: 22 August 1995  相似文献   

5.
Production of hydrolytic enzymes by oral isolates of Eikenella corrodens   总被引:3,自引:0,他引:3  
Abstract Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65°C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in T. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.  相似文献   

6.
Several proteases, including the bacterial serine protease subtilisins, require the assistance of the N-terminal pro-sequence of precursors to produce active, mature enzymes. Upon completion of folding, the pro-sequence is autocatalytically degraded because it is not necessary for the activity or stability of folded, mature cognates of the original enzymes. Therefore, the pro-sequence functions as an intramolecular chaperone that guides correct folding of the protease domain. Interestingly, Shinde et al. proposed a new theory of "protein memory" in which an identical polypeptide can fold into an altered conformation with different secondary structure, stability and specificities through a mutated pro-sequence [Shinde et al. (1997) Nature 389:520–522]. We also showed that the autoprocessing efficiency was improved by modifications in the pro-sequence of mutant subtilisins with altered substrate specificity. Further, the pro-sequence from a subtilisin homologue was found to chaperone the intramolecular folding of denatured subtilisin. These results indicate that engineering of the pro-sequence, i.e., site-directed and/or random mutagenesis, chimeras and gene shuffling between members of the family, would be a useful method for improving the functions of autoprocessing proteases. Conventional protein engineering techniques have thus far employed mutagenesis in the protease domain to modify the enzymatic properties. This new approach, which we term "pro-sequence engineering", is not only an important tool for studying the mechanism of protein folding, but also a promising technology for creating unique proteases with various beneficial properties.  相似文献   

7.
An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Delta) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 degrees C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 degrees C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNDeltaC105. By analyzing the product of the pMAQ-c2Delta-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.  相似文献   

8.
An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Δ) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 °C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 °C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNΔC105. By analyzing the product of the pMAQ-c2Δ-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.  相似文献   

9.
Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.  相似文献   

10.
The Bacillus cereus cnp gene coding for the thermolysin-like neutral protease (TNP) has been cloned, sequenced, and expressed in Bacillus subtilis. The protease is first produced as a pre-pro-protein (M(r) = 61,000); the pro-peptide is approximately two-thirds of the size of the mature protein. The pro-sequence has been compared with those of six other TNPs, and significant homologies have been found. Additionally, the TNP pro-sequences are shown to be homologous to the pro-sequence of Pseudomonas aeruginosa elastase. A mutant has been constructed from cnp, in which 23 amino acids upstream from the pro-protein processing site have been deleted. This region has no homologous analogue in any of the other TNP pro-sequences. The deletion results in a delay of six to eight hours in detection of active protease in the growth medium, as well as a 75% decrease in maximum protease production. N-terminal analysis of the mutant mature protein demonstrates that the processing site is unaltered by the pro-sequence deletion. The deletion must, therefore, modulate the kinetics of processing and/or secretion of the pro-protein.  相似文献   

11.
Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.  相似文献   

12.
Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.  相似文献   

13.
Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.  相似文献   

14.
The cysteine endopeptidase streptopain, an extracellular enzyme from pathogenic Streptococcus pyogenes, is synthesized as a precursor containing an NH2-terminal pro-sequence. The pro-sequence of streptopain was expressed in Escherichia coli and subjected to structural and functional investigation. Heat-induced denaturation of the pro-sequence studied using circular dichroism spectroscopy revealed that it forms a compact structure and represents an independently folded domain. The isolated pro-sequence exhibits high affinity towards mature streptopain and associates with its cognate enzyme by forming an equimolar complex. Refolding of denatured streptopain in the presence of pro-sequence in vitro facilitated recovery of active enzyme. Expression of the mature streptopain in E. coli either alone, or in trans with its pro-sequence as an independent polypeptide, led to the formation of insoluble protein aggregates or functionally active enzyme, respectively. These results demonstrate that the pro-sequence domain acts as an intramolecular chaperone that directs the correct folding of the mature streptopain.  相似文献   

15.
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.  相似文献   

16.
Subtilisin E, an alkaline serine protease of Bacillus subtilis 168, is first produced as a precursor, pre-pro-subtilisin, which consists of a signal peptide for protein secretion (pre-sequence) and a peptide extension of 77 amino acid residues (pro-sequence) between the signal peptide and mature subtilisin. When the entire coding region for pre-pro-subtilisin E was cloned into an Escherichia coli expression vector, active mature subtilisin E was secreted into the periplasmic space. When the pre-sequence was replaced with the E. coli OmpA signal peptide, active subtilisin E was also produced. When the OmpA signal peptide was directly fused to the mature subtilisin sequence, no protease activity was detected, although this product had the identical primary structure as subtilisin E as a result of cleavage of the OmpA signal peptide and was produced at a level of approximately 10% of total cellular protein. When the OmpA signal peptide was fused to the 15th or 44th amino acid residue from the amino terminus of the pro-sequence, active subtilisin was also not produced. These results indicate that the pro-sequence of pre-pro-subtilisin plays an important role in the formation of enzymatically active subtilisin. It is proposed that the pro-sequence is essential for guiding appropriate folding of the enzymatically active conformation of subtilisin E.  相似文献   

17.
Der fI is a cysteine protease contained in feces of mites and is one of major mite allergens. Recombinant Der fI (reDer fI) that is produced using a baculovirus expression system contains pro-sequences of different lengths. Most of these can be removed by acid treatment. However, IgE-binding activity of acid-treated reDer fI is lower than that of native Der fI at high protein concentrations, and N-terminal amino acids of acid-treated reDer fI are not uniform. Now, a method for processing of the pro-sequence has been developed by producing reDer fI E(?1)K with baculovirus expression system in which the carboxy terminal amino acid of the pro-sequence (glutamate) was replaced by lysine using site directed mutagenesis. No difference in the amount of production was observed upon introducing the mutation into the pro-sequence. Addition of lysylendopeptidase into the culture medium led to processing of the pro-sequence of reDer fI E(?1)K and proceeded the degradation of the other proteins in the medium. Lysylendopeptidase-treated reDer fI E(?1)K was easily purified with an anion exchange column, resulting in 20% increase of the yield. Lysylendopeptidase-treated reDer fI E(?1)K obtained through these processes was compared with the native Der fI. Although some differences were found in protease activity and reactivity with lectins, their N-terminal amino acid and the IgE-binding activity were the same as those of the native one, indicating its usefulness for diagnostic purpose.  相似文献   

18.
We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.  相似文献   

19.
20.
Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25-28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2-terminal sequence previously reported and the determined amino acid sequences, including the COOH-terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28,350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin-type serine proteases, and 43% identity with proteinase K, 37-39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2-terminal portion, the protease and a COOH-terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68-74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.  相似文献   

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