Expression of Eukaryotic Genes in B. subtilis Using Signals of penP

Chang, Shing; Gray, O; Ho, Diana; Kroyer, James; Chang, Sheng-Yung; McLaughlin, Jane R.; Mark, D.

doi:10.1016/b978-0-12-274150-0.50021-7

Cited by 13 publications

(16 citation statements)

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“…We have independently cloned penP and the repressor gene penI from both the wild-type and constitutive strains of B. licheniformis 9945A (14). By use of the penP signal sequence, many protein secretion vectors have been constructed in Bacillus subtilis (3,10,13).…”

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Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Himeno¹,

Imanaka²,

Aiba³

1986

J Bacteriol

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Bacillus licheniformis penicillinase genes, penP and penl, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penl(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48°C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB6OE24), the coding region was concluded to be the pen! gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penl promoter. The regulation of penP and pen! by the repressor is discussed.

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Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Himeno¹,

Imanaka²,

Aiba³

1986

J Bacteriol

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“…5, the estimated half-life of P-lactamase in B. subtilis 168(pWB), DB428(pWB), WB500(pWB), WB600 (pWB), WB600(pQL), and WB600(pYL) was 1.5, 21, 27, 41, FIG. 5. Stability of the secreted P-lactamase in the culture of the wild-type B. subtilis 168(pWB) (+), WB600(pQL) (0), DB428(pWB) (A), WB500(pWB) (0), WB600(pYL) (A), and WB600(pWB) (O).…”

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confidence: 99%

“…Although degQ can stimulate the production of P-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.With the capability of secreting extracellular enzymes directly into the culture medium, Bacillus subtilis potentially can serve as an efficient expression host (4,5,9,24,25). The secreted foreign proteins usually remain in biologically active forms (20,21), and the downstream purification is greatly simplified.…”

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“…However, at least two major limitations hinder the wide application of B. subtilis as a production microorganism. First, B. subtilis produces and secretes high levels of extracellular proteases which degrade the secreted foreign proteins (5,41). It is well established that B. subtilis has six extracellular proteases: neutral protease A (45), subtilisin (also known as alkaline protease) (34,42), extracellular protease (29,37), metalloprotease (30), bacillopeptidase F (31,43), and neutral protease B (36).…”

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Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases

et al. 1991

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We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extraceilular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride.By using TEM P-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in ,-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of P-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.With the capability of secreting extracellular enzymes directly into the culture medium, Bacillus subtilis potentially can serve as an efficient expression host (4,5,9,24,25). The secreted foreign proteins usually remain in biologically active forms (20,21), and the downstream purification is greatly simplified. However, at least two major limitations hinder the wide application of B. subtilis as a production microorganism. First, B. subtilis produces and secretes high levels of extracellular proteases which degrade the secreted foreign proteins (5, 41). It is well established that B. subtilis has six extracellular proteases: neutral protease A (45), subtilisin (also known as alkaline protease) (34, 42), extracellular protease (29,37), metalloprotease (30), bacillopeptidase F (31, 43), and neutral protease B (36). The construction of a strain deficient in five proteases GP263 (30,32), has eliminated over 99% of total extracellular protease activity. However, depending on the nature of the foreign proteins to be expressed in this strain, some of them remain unstable. Second, the lack of well-regulated inducible vectors also limits the wide application of the B. subtilis system. To overcome this problem, a few inducible vectors have been developed. Most of these vectors use either the E. coli lac system (16,46) or the temperature-sensitive repressor from lambda (3) or 4105 (23) to regulate the expression of foreign genes. Some others (10,39,47,48) are based on the regulatory region of a B. ...

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“…This bottleneck reduces the application efficacy. Bacillus subtilis can serve as an efficient and safe host for recombinant protein secretion (3,4,7,16,17). Secreted proteins usually remain in biologically active forms (13,15), and downstream purification is greatly simplified.…”

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High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800

Zhang

Cui

Hong

et al. 2005

Appl Environ Microbiol

148

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The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.Organophosphate compounds are used extensively in agricultural and domestic pest control and as chemical warfare agents; these compounds inhibit the acetylcholinesterase of animals (8). Mass application of these compounds in the environment causes serious problems. Organophosphate hydrolase (OPH) isolated from soil microorganisms detoxified organophosphates effectively (14,18), and bioremediation of organophosphate compounds in the environment by using bacterial enzymes may provide an efficient, convenient, and economical method for detoxification. We previously isolated Plesiomonas sp. strain M6, which is capable of hydrolyzing methyl parathion at high efficiency (5), and the gene (mpd) encoding a novel methyl parathion hydrolase (MPH) was cloned (5) and expressed in Escherichia coli (9). However, the previous work showed that E. coli could process only a small proportion of the inactive precursor polypeptide, comprising the signal peptide and the mature enzyme, to produce the active MPH (9).Most microorganisms that produce OPH are gram-negative bacteria, and the OPH is located intracellularly or secreted into the periplasm. The outer membrane acts as a permeability barrier and limits interaction between the pesticides and OPH residing within the cells (12). This bottleneck reduces the application efficacy. Bacillus subtilis can serve as an efficient and safe host for recombinant protein secretion (3,4,7,16,17). Secreted proteins usually remain in biologically active forms (13,15), and downstream purification is greatly simplified. In this work, the mpd gene was fused with the nprB signal peptideencoding sequence, and the organophosphate hydrolase was expressed and secreted in B. subtilis under the control of the P43 promoter (20).The methyl parathion hydrolase-encoding gene mpd (GenBank accession no. AF338729) cannot be expressed in B. subtilis by using its own promoter. To achieve expression and secretion of MPH in B. subtilis, the secretion signal peptideencoding sequence of the nprB gene was fused with the mature mpd gene. A typical cleavage site (ASA-A) (19) for signal peptidase I was designed for the release from B. subtilis of MPH with an authentic N terminus (Fig. 1B). The mature mpd gene sequence (9) was cloned using the primer pair P1 (5Ј-G CGCTGCAGCACCGCAGGTG-3Ј) and P2 (5Ј-CGCAAGCT TTCATCATCACTTGGGGTTGACGACCGA-3Ј) from plasmid pMT1 (5). To introduce the PstI site (underlined) upstream, the codon GCC encoding the first amino acid of mature MPH was modified to GCA (Fig. 1B). Two additional stop codons (TGATGA) and the HindIII site (underlined) were introduced with primer P2. Primers P3 (5Ј-CGCGGAT CCTGATAGGTGGTATGTTTTCGC-3Ј) and P4 (5Ј-CTTG GTCAAGTTGCGCATGTGTACATTCCTCTCTT-3Ј) were used to ...

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Expression of Eukaryotic Genes in B. subtilis Using Signals of penP

Cited by 13 publications

References 15 publications

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases

High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800

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