Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor.

Yamagata, Hideo; Nakahama, Kazuo; Suzuki, Yutaka; Kakinuma, Atsushi; Tsukagoshi, Norihiro; Udaka, Shigezo

doi:10.1073/pnas.86.10.3589

Cited by 97 publications

(58 citation statements)

References 39 publications

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“…If the E. coli ␣-hemolysin experience is an indicator, it should be possible to link a wide variety of proteins to the RsaA secretion signal and achieve high-level secretion of hybrid proteins into the growth medium. So far, only the S-layer protein of Bacillus brevis has been exploited for heterologous protein secretion; the secretion of this protein relies on a classical N-terminal signal leader peptide (64).…”

Section: Discussionmentioning

confidence: 99%

Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion

1997

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Linker insertion mutagenesis was used to modify the paracrystalline surface layer (S-layer) protein (RsaA) of the gram-negative bacterium Caulobacter crescentus. Eleven unique BamHI linker insertions in the cloned rsaA gene were identified; at the protein level, these linker insertions introduced 4 to 6 amino acids at positions ranging from the extreme N terminus to the extreme C terminus of the 1,026-amino-acid RsaA protein. All linker-peptide insertions in the RsaA N terminus caused the secreted protein to be shed into the growth medium, suggesting that the RsaA N terminus is involved in cell surface anchoring. One linker-peptide insertion in the RsaA C terminus (amino acid 784) had no effect on S-layer biogenesis, while another (amino acid 907) disrupted secretion of the protein, suggesting that RsaA possesses a secretion signal lying C terminal to amino acid 784, near or including amino acid 907. Unlike extreme N-or C-terminal linker-peptide insertions, those more centrally located in the RsaA primary sequence had no apparent effect on S-layer biogenesis. By using a newly introduced linker-encoded restriction site, a 3 fragment of the rsaA gene encoding the last 242 C-terminal amino acids of the S-layer protein was expressed in C. crescentus from heterologous Escherichia coli lacZ transcription and translation initiation information. This C-terminal portion of RsaA was secreted into the growth medium, confirming the presence of a C-terminal secretion signal. The use of the RsaA C terminus for the secretion of heterologous proteins in C. crescentus was explored by fusing 109 amino acids of an envelope glycoprotein from infectious hematopoietic necrosis virus, a pathogen of salmonid fish, to the last 242 amino acids of the RsaA C terminus. The resulting hybrid protein was successfully secreted into the growth medium and accounted for 10% of total protein in a stationary-phase culture. Based on these results and features of the RsaA primary sequence, we propose that the C. crescentus S-layer protein is secreted by a type I secretion system, relying on a stable C-terminal secretion signal in a manner analogous to E. coli ␣-hemolysin, the first example of an S-layer protein secreted by such a pathway.The outer membrane of the dimorphic gram-negative bacterium Caulobacter crescentus is covered by a protein surface layer (S-layer) composed of a single 1,026-amino-acid protein termed RsaA (13). The S-layer is paracrystalline in nature, exhibiting an array of ring-like subunits (each composed of six copies of RsaA) arranged on a lattice with p6 symmetry and interlinked at the threefold rotational axis (57); proper crystallization of the S-layer is dependent on Ca 2ϩ ions (62, 63). The association between RsaA and the outer membrane is not completely understood, but the protein appears to be anchored to the outer membrane via noncovalent interactions with a specific smooth lipopolysaccharide (LPS) molecule (62). So far, the only known function of the C. crescentus S-layer is to protect cells against predation by a Bde...

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Section: Discussionmentioning

confidence: 99%

Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion

1997

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“…By using this system, several proteins with multiple disulfide bonds and the same activity as native proteins can be efficiently produced (34). Bdb may play an important role in the disulfide bond formation of these heterologous proteins on the cell surface of B. brevis.…”

Section: Discussionmentioning

confidence: 99%

Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in Bacillus brevis

et al. 1995

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The gene (bdb) for protein thiol-disulfide oxidoreductase cloned from Bacillus brevis was found to encode a polypeptide consisting of 117 amino acid residues with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of disulfide oxidoreductases such as DsbA, protein disulfide isomerase, and thioredoxin. The deduced amino acid sequence showed significant homology with those of several bacterial thioredoxins. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B. brevis was enzymatically active in both reduction and oxidization of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.Disulfide bonds play an important role in producing the three-dimensional structures responsible for the specific properties of proteins. Although disulfide bonds in a protein can form spontaneously in vitro, the process is much slower and less effective than that in vivo (20), suggesting the existence of proteins which catalyze native disulfide bond formation in vivo. Disulfide oxidoreductases such as thioredoxin and protein disulfide isomerase (PDI) contain the Cys-X-X-Cys motif as the catalytic active site (16) and facilitate disulfide exchange in vitro (10).The dsbA gene in Escherichia coli has been discovered independently by two groups (3, 12). DsbA is a periplasmic protein with the sequence motif Cys-Pro-His-Cys, which resembles the active site of thioredoxin and PDI. The dsbA mutation causes a defect in the disulfide bond formation of several periplasmic proteins such as alkaline phosphatase, the OmpA protein, ␤-lactamase (3), and the P-ring protein of the flagellar basal body (7). DsbA appears to be required for the formation of disulfide bonds. DsbA homologs are also found in bacteria such as Vibrio cholerae (19) and Haemophilus influenzae (26). They are thought to form a new family of bacterial disulfide oxidoreductase.All bacterial disulfide oxidoreductases have been isolated only from gram-negative bacteria, while no data have been obtained on those of gram-positive bacteria. In gram-positive bacteria, secretory nascent polypeptides are transported to the external medium and must fold into precise shapes to produce functional proteins outside or on the surface of the cell. It is of interest to examine how extracellular proteins acquire disulfide bonds in the protein secretory pathway. We describe here the cloning and characterization of a gene termed bdb (for Bacillus disulfide bond formation), which encodes the protein thioldisulfide oxidoreductase of Bacillus brevis, a gram-positive bacterium used as a host for heterologous protein production (31). MATERIALS AND METHODSBacterial strains, media, plasmids, and transformation. The bacterial strains used were B. brevis HPD31 (24) and E. coli XL1-Blue (Stratagene), JCB502 ( D69 lacZ::Tn10 tetS by fusaric acid) (3), JCB572 (JCB502 dsbA::kan-1) (...

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“…A major drawback of the fusion approach is the formation of rEGF as variants possessing different peptide lengths (Table 1) [11][12][13][14][15][16][17][18][19][20][21][22]. Moreover, the EGF derivatives were commonly shown to exhibit lower levels of bioactivity and stability [17,[22][23][24].…”

Section: Approaches Of Expressing Recombinant Egfmentioning

confidence: 99%

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2017

JAPLR

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Human epidermal growth factor (EGF) is a functionally versatile polypeptide comprising 53 amino acids (aa). Due to the ability of EGF to stimulate epidermal growth and proliferation, numerous research studies have been conducted to explore its potential applications in cosmetic, skin care and medication. The emergence of recombinant DNA technology has facilitated the production of a great variety of recombinant EGF (rEGF) isoforms. However, increasing evidence supports that rEGF isoforms exhibit different levels of stability and potency. This review discusses how the less bioactive rEGF isoforms may mystify the identity and even the functional properties of authentic rEGF, which shares the same primary structure as native EGF. The identity crisis, the time-consuming, costly and labor-intensive patenting and drug regulatory processes act together to result in underrating the functional applications of authentic rEGF, which have been shown to be effective and safe in promoting treatments of a variety of skin disorders such as diabetic foot ulcers (DFU).

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Use of Bacillus brevis for efficient synthesis and secretion of human epidermal growth factor.

Cited by 97 publications

References 39 publications

Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion

Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion

Cloning and characterization of the gene for a protein thiol-disulfide oxidoreductase in Bacillus brevis

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