Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable  -amylases in Bacillus subtilis and Escherichia coli cells

Itoh, Yoshiki; Kanoh, K.; Nakamura, Kouji; Takase, Kazuma; Yamane, Kunio

doi:10.1099/00221287-136-8-1551

Cited by 11 publications

(5 citation statements)

References 30 publications

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“…This improvement was accompanied by a two-to fourfold increase in the amounts of Nuc-secreted product (up to 20 mg per liter). Similar experiments in which several synthetic peptides were inserted just after the signal peptide in a heterologous hybrid protein in B. subtilis resulted in slightly enhanced secretion; however, processing occurred at an altered cleavage site (14). In contrast to the results of those previous studies, LEISSTCDA-Nuc fusions conserve the original cleavage site.…”

Section: Discussionmentioning

confidence: 47%

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Loir¹,

Gruss²,

Ehrlich

et al. 1998

J Bacteriol

166

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Lactococcus lactis, a gram-positive organism widely used in the food industry, is a potential candidate for the secretion of biologically useful proteins. We examined the secretion efficiency and capacity of L. lactis by using the Staphylococcus aureus nuclease (Nuc) as a heterologous model protein. When expressed in L. lactis from an efficient lactococcal promoter and its native signal peptide, only ∼60% of total Nuc was present in a secreted form at ∼5 mg per liter. The remaining 40% was found in a cell-associated precursor form. The secretion efficiency was reduced further to ∼30% by the deletion of 17 residues of the Nuc native propeptide (resulting in NucT). We identified a modification which improved secretion efficiency of both native Nuc and NucT. A 9-residue synthetic propeptide, LEISSTCDA, which adds two negative charges at the +2 and +8 positions, was fused immediately after the signal peptide cleavage site. In the case of Nuc, secretion efficiency was increased to ∼80% by LEISSTCDA insertion without altering the signal peptide cleavage site, and the yield was increased two- to fourfold (up to ∼20 mg per liter). The improvement of NucT secretion efficiency was even more marked and rose from 30 to 90%. Similarly, the secretion efficiency of a third protein, the α-amylase ofBacillus stearothermophilus, was also improved by LEISSTCDA. These data indicate that the LEISSTCDA synthetic propeptide improves secretion of different heterologous proteins in L. lactis.

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Section: Discussionmentioning

confidence: 47%

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Loir¹,

Gruss²,

Ehrlich

et al. 1998

J Bacteriol

166

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“…Instead, we believe that they are removed by the several nonspecific proteases secreted by Bacillus species. This mechanism is supported by the facts that artificial propeptides can also be accurately removed (139), as can natural propeptides when fused to such proteins that do not normally contain them (319). Furthermore, when B. licheniformis 3-lactamase was produced in a B. subtilis mutant with low exoprotease activity, a much greater percentage of the enzyme remained cell associated at late growth phases than that in wild-type B. subtilis (245).…”

Section: Propeptidesmentioning

confidence: 99%

“…The different lengths of signal peptides may be related to differences in the structure of signal peptidases or other secretion components in gram-positive and gram-negative bacteria. For example, the signal peptidases of E. coli and Bacillus species often cleave the same signal peptides at different sites, E. coli favoring cleavage sites that produce shorter signal peptides than those of Bacillus species (139,284,332,342).…”

Section: Signal Peptidesmentioning

confidence: 99%

“…In fact, exact fusions are sometimes more efficiently processed than natural, preserved sites (110,113,232,249,296). It appears from several different experiments that the sequences beyond the cleavage site can have a remarkable effect on the export and production efficiencies (110,113,139,296). Preservation of the surroundings of the cleavage site results in vector-derived NH2-terminal residues in the secreted protein.…”

Section: Foreign Proteinsmentioning

confidence: 99%

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Protein secretion in Bacillus species.

Simonen¹,

Palva²

1993

Microbiological Reviews

239

146

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During recent years new data have accumulated, greatly improving our understanding of the export processes in bacteria, yeasts, and mammalian cells. The purpose of this article is to describe the present state of knowledge of protein export in Bacillus species, although it is still relatively poorly characterized. Therefore we first describe the more thoroughly characterized export systems of Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. General Exported proteins are synthesized initially as preproteins with an amino-terminal extension, the signal peptide.

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“…Signal peptide cleavage sites were determined by the NH2-terminal amino acid sequences of the exported thermostable a-amylases from the hybrid genes. It has been shown that a possible secondary proteolysis in the a-amylase preparations did not occur or was negligible under the present culture conditions with the protease-deficient B. subtilis strain and E. coli HB101 as the host cells and during the subsequent purification steps under hydrophobic conditions (11,12 subtilis. (b) Nucleotide and predicted amino acid sequences around the junction regions of B. subtilis ax-amylase signal peptide (amyE') and the mature thermostable a-amylase ('amyT) of B. stearothermophilus A631 in plasmids pTUBE691, pTUBE693, and pTUBE695, in which one, two, and three Ala-X-Ala sequences were inserted, respectively.…”

mentioning

confidence: 92%

Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides

et al. 1993

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The Bacilus subtilis ai-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis a-amylase signal peptides of different lengths and the mature thermostable a-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable at-amylases and by in vivo pulse-chase experiments. In B. subfilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and ca-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.Most exported proteins are synthesized as precursors with an NH2-terminal extension, the signal peptide, which is removed by a specific enzyme. The signal peptide and signal peptidase mechanisms for the secretion of exported proteins are similar in both prokaryotic and eukaryotic cells (2,8,10,22). In prokaryotic cells, the amino acid residues at positions -3 and -1 are important for cleavage site recognition by the signal peptidases when signal peptides are cleaved between positions -1 and + 1, with position -1 being particularly important (30). On the basis of the amino acid sequences of signal peptides reported by Watson (31) and Perlman and Halvorson (23), we compared the amino acid residues at positions -3 and -1 in 34 exported proteins of prokaryotic cells, excluding lipoproteins, whose signal peptides are cleaved by signal peptidase II (6). Eighty percent (27 of 34) of the amino acid residues at position -1 consisted of Ala, 44% (15 of 34) of the amino acid sequences at positions -3 and -1 were Ala-X-Ala, and 9% (3 of 34) of the sequences were Val-X-Ala and Thr-X-Ala. The sequence Ala-X-Ala predominates at positions -3 and -1 of the signal peptide cleavage site in precursors of exported proteins.Yamazaki et al. (34) predicted the amino acid sequence around the signal peptide cleavage site of Bacillus subtilis a-amylase to be Gly-27-Pro-Ala-Ala-Ala-Ser-Ala I GluThr-35. The cleavage site was confirmed as the peptide bond between Ala-33 and Glu-34 (Fig. la) In the signal peptide of AmyE, there were two Ala-X-Ala sequences at positions 29 to 31 and 31 to 33 near the signal peptide cleavage site. A cleavage s...

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Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable -amylases in Bacillus subtilis and Escherichia coli cells

Cited by 11 publications

References 30 publications

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

A Nine-Residue Synthetic Propeptide Enhances Secretion Efficiency of Heterologous Proteins in Lactococcus lactis

Protein secretion in Bacillus species.

Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides

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