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

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.     

Secretion activities of Bacillus subtilis alpha-amylase signal peptides of different lengths in Escherichia coli cells

The B. subtilis alpha-amylase promoter and signal peptide are functional in E. coli cells. DNA fragments coding for signal peptides with different lengths (28, 31, 33 and 41 amino acids from the translation initiator Met) were prepared and fused with the E. coli beta-lactamase structural gene. In B. subtilis cells, the sequences of 31, 33 and 41 amino acids were able to secrete beta-lactamase into the surrounding media, but the 28 amino acid sequence was not. In contrast, all of the four sequences were able to export beta-lactamase into the periplasmic space of E. coli cells. Thus, the recognition of the B. subtilis alpha-amylase signal peptide in E. coli cells seems to be different from that in B. subtilis cells.     

Use of alkaline phosphatase fusions to study protein secretion in Bacillus subtilis.

We have constructed a vector designed to facilitate the study of protein secretion in Bacillus subtilis. This vector is based on a translational fusion between the expression elements and signal sequence of Bacillus amyloliquefaciens alkaline protease and the mature coding sequence for Escherichia coli alkaline phosphatase (phoA). We show that export of alkaline phosphatase from B. subtilis depends on a functional signal sequence and that alkaline phosphatase activity depends upon secretion. The vector design facilitates the insertion of heterologous coding sequences between the signal and phoA to generate three-part translational fusions. Such phoA fusions are easily analyzed by monitoring alkaline phosphatase activity on agar plates or in culture supernatants or by immunological detection. Exploitation of this methodology, which has proven to be extremely useful in the study of protein secretion in E. coli, has a variety of applications for studying protein secretion in B. subtilis.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

Length and structural effect of signal peptides derived from Bacillus subtilis alpha-amylase on secretion of Escherichia coli beta-lactamase in B. subtilis cells.

The precursor of Bacillus subtilis alpha-amylase contains an NH2-terminal extension of 41 amino acid residues as the signal sequence. The E. coli beta-lactamase structural gene was fused with the DNA for the promoter and signal sequence regions. Activity of beta-lactamase was expressed and more than 95% of the activity was secreted into the culture medium. DNA fragments coding for short signal sequences 28, 31, and 33 amino acids from the initiator Met were prepared and fused with the beta-lactamase structural gene. The sequences of 31 and 33 amino acid residues with Ala COOH-terminal amino acid were able to secrete active beta-lactamase from B. subtilis cells. However beta-lactamase was not secreted into the culture medium by the shorter signal sequence of 28 amino acid residues, which was not cleaved. Molecular weight analysis of the extracellular and cell-bound beta-lactamase suggested that the signal peptide of B. subtilis alpha-amylase was the first 31 amino acids from the initiator Met. The significance of these results was discussed in relation to the predicted secondary structure of the signal sequences.     

A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).     

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the alpha-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments. Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E. coli. Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.     

Secretion of human interferon-alpha induced by using secretion vectors containing a promoter and signal sequence of alkaline phosphatase gene of Escherichia coli

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.     

来源于Bacillus subtilis的中性植酸酶基因的克隆及在大肠杆菌中的表达

通过ＰＣＲ的方法从Bacillus subtilis基因组中克隆了中性植酸酶基因nphy,ＤＮＡ全序列分析表明其结构基因全长１１５２个核苷酸（编码３８３个氨基酸）,５′端有一编码２６个氨基酸的信号肽序列。去除信号肽编码序列的nphy克隆到大肠杆菌ＩＰＴＧ诱导表达载体pTYB40上,在大肠杆菌中得到了高效表达,表达量达到大肠杆菌可溶性蛋白的４０％以上,表达产物具有生物学活性,证实了克隆到的中性植酸酶的基因有正常的生物学功能。     

Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus

Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH2-terminal end. These regions may be involved in substrate binding and/or catalytic sites.     

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrase gene (scrB)

The nucleotide sequence of a 2.119-kb DNA fragment containing the Vibrio alginolyticus sucrase gene (scrB) was determined. The complete sequence (484 aa residues) of the sucrase was deduced and homology was detected between the sucrase enzymes from V. alginolyticus and the Gram-positive bacteria Bacillus subtilis and Streptococcus mutans. In Escherichia coli cells the cloned V. alginolyticus sucrase is translocated to the periplasm. Transposon phoA mutagenesis experiments strongly suggested that V. alginolyticus sucrase in E. coli is not exported across the cytoplasmic membrane by means of a typical signal sequence.     

Expression of Bacillus amyloliquefaciens amylase and Vibrio alginolyticus protease A fusion genes.

Previously we reported [Deane, S. M., Maharaj, R., Robb, F. T. & Woods, D. R. (1987) Journal of General Microbiology 133, 2295-2302] that the production of a Vibrio alginolyticus SDS-resistant alkaline serine protease (Pro A) cloned in Escherichia coli was characterized by a 12 h delay between the synthesis of an inactive precursor and secretion of active Pro A. Replacement of the V. alginolyticus promoter region by the alpha-amylase promoter region from Bacillus amyloliquefaciens resulted in the simultaneous synthesis and secretion of Pro A in E. coli. The V. alginolyticus pro A gene cloned on a shuttle vector did not produce active Pro A in Bacillus subtilis. Although Pro A has a typical Gram-positive signal sequence, it was not functional in B. subtilis. Replacement of the Pro A signal sequence with the alpha-amylase signal sequence resulted in the production of active Pro A in B. subtilis.     

地衣芽孢杆菌α-淀粉酶基因的克隆和及其启动子功能鉴定

根据已知α-淀粉酶编码基因保守区核苷酸序列,通过PCR和反向PCR技术克隆出Bacillus licheniformisCICIM B0204α-淀粉酶编码基因amyL全长序列及其上下游序列。B.licheniformisCICIM B0204amyL由1539bp组成,其上游180bp为启动子序列,下游160bp为终止子序列;成熟肽由512个氨基酸残基组成,氨基端的29个氨基酸残基为α-淀粉酶的信号肽。通过基因及其氨基酸序列比对发现,amyL及其编码产物与芽孢杆菌来源的α-淀粉酶具有高度相似性。将amyL的结构基因在PT7介导下于大肠杆菌中诱导表达,获得具有α-淀粉酶活性的表达产物。将amyL的启动子序列和信号肽序列与B.licheniformisCICIM B2004的β-甘露聚糖酶结构基因进行读框内重组,在大肠杆菌中获得了β-甘露聚糖酶的分泌表达,重组大肠杆菌表达295U/mL的β-甘露聚糖酶酶活。     

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.     

Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.