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.     

Proinsulin cDNAs from the leopard frog, Rana pipiens: evolution of proinsulin processing

We have isolated a proinsulin cDNA from the Amphibian Rana pipiens. The predicted R. pipiens insulin A- and B-chain amino acid sequences differ from that deduced from the closely related Rana catesbeiana at one residue (Asp for Pro at B2). The R. pipiens and Xenopus laevis proinsulin precursor sequences are of identical length, with the amino acid sequences of the mature A- and B-chains being well conserved. The proinsulin C-peptide amino acid sequence is less well conserved between R. pipiens and X. laevis and also differs in length. The R. pipiens C-peptide is shorter than the homologous X. laevis sequence due to a two amino acid residue truncation. The truncation of the R. pipiens C-peptide compensates for a two amino acid residue extension observed at the N-terminal of the A-chains of insulins from Ranid frogs. A change in the site of proinsulin processing can explain both the C-peptide and A-chain length differences. The evolution of the new proinsulin processing site required two amino acid substitutions.     

Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor

Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions. Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4-24 mM) to Schaeffer's medium containing glucose (4 g l(-1)). If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors. Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures. Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate. In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.     

Isolation and characterization of a genetic variant of bovine proinsulin

A genetic variant of bovine proinsulin has been isolated using preparative reverse-phase HPLC. The new proinsulin (bovine proinsulin II) differs from the known proinsulin (bovine proinsulin I) by a single amino acid residue at position C-48 in the connecting peptide. The amino acid replacement is a leucine substitution for proline. The two proinsulins were found in a ratio of approximately 9:1, proinsulin I: proinsulin II. No chemical or biological differences were observed for the two proinsulins other than their different elution times on reverse-phase HPLC.     

Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjang and its expression in protease-deficient Bacillus subtilis strains

Bacillus subtilis CH3-5 was isolated from cheonggukjang prepared according to traditional methods. CH3-5 secreted at least four different fibrinolytic proteases (63, 47, 29, and 20 kDa) into the culture medium. A fibrinolytic enzyme gene, aprE2, encoding a 29 kDa enzyme was cloned from the genomic DNA of CH3-5, and the DNA sequence determined. aprE2 was overexpressed in heterologous B. subtilis strains deficient in extracellular proteases using a E. coli-Bacillus shuttle vector. A 29 kDa AprE2 band was observed and AprE2 seemed to exhibit higher activities towards fibrin rather than casein.     

Antihypertensive Effect of Boysenberry Seed Polyphenols on Spontaneously Hypertensive Rats and Identification of Orally Absorbable Proanthocyanidins with Vasorelaxant Activity

An E. coli expression clone coding for human proinsulin, which was fused to NH₂-terminal β-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH₂ terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)_n, (His)_n, (Trp)_n, and (Ser)_n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH₂-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.     

Secretion of correctly processed and folded pancreatic secretory trypsin inhibitor by Bacillus subtilis.

We constructed a plasmid, designated pNPP126, containing a DNA sequence encoding a fusion protein composed of Bacillus amyloliquefaciens neutral protease prepeptide (signal peptide) and human pancreatic secretory trypsin inhibitor (hPSTI), where the mature hPSTI is accurately fused to the 3'-terminal of the prepeptide coding region. It was observed that the strain Bacillus subtilis MT600 harboring pNPP126 could secrete a trypsin inhibitory activity into the culture medium. The N-terminal amino acid sequence, the amino acid composition and the stoichiometry of the purified hPSTI produced by B. subtilis were the same as those of natural hPSTI, indicating that the transformant B. subtilis MT600 (pNPP126) could efficiently secrete the correctly processed and folded hPSTI into the culture medium.     

基因工程人胰岛素原和胰岛素的分离纯化及性质研究

E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.     

利用枯草杆菌碱性蛋白酶E基因的信号顺序构建分泌表达载体

本文利用枯草杆菌碱性蛋白酶基因Ｅ（ａｐｒＥ）,对建立枯草杆菌分泌表达的载体－宿主系统作了探讨。首先用大肠杆菌－枯草杆菌穿梭的启动子克隆质粒ｐＧＫＶ２１０对ａｐｒＥ的启动子功能进行检测,发现用Ｅ．ｃｏｌｉ作为研究ａｐｒＥ表达信号的“中间宿主”是可行的;然后在穿梭质ｃｏｌｉ作为研究,ａｐｒＥ表达信号的“中间宿主”是可行的;然后在穿梭质粒,进而构建了基于ａｐｒＥ启动子和信号顺序的分泌表达载体ｐＳＰ１和ｐ     

Cloning and overexpression of <Emphasis Type="Italic">aprE3-17</Emphasis> encoding the major fibrinolytic protease of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> CH 3-17

Bacillus licheniformis (B. licheniformis) CH3-17, an isolate from cheonggukjang, a traditional Korean fermented soyfood, secretes several fibrinolytic enzymes into the culture medium, showing strong fibrinolytic activity. A gene homologous to aprE of Bacillus subtilis (B. subtilis), aprE3-17, was cloned by PCR. DNA sequencing showed that aprE3-17 encodes a prepro-type serine protease consisting of 382 amino acids. The mature enzyme was 27 kDa in size. The aprE3-17 gene was overexpressed in B. subtilis WB600 using pHY300PLK, an Escherichia coli (E. coli)-Bacillus shuttle vector, and the 27 kDa enzyme was purified from the culture supernatant. The optimum pH for activity was 6.0. Purified enzyme quickly degraded the Aα and Bβ chains of fibrinogen but could not degrade the γ-chain.     

Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

Analytical biotechnology of recombinant peptides and proteins. II. Primary structure of the fusion protein containing human proinsulin and optimization of its proteolysis by trypsin

The kinetics of trypsin proteolysis of the fusion protein (FP) containing human proinsulin was studied by a set of analytical micromethods. These were the microcolumn reversed-phase HPLC and the qualitative identification by MALDI-TOF mass spectrometry and amino acid sequencing. The first stage of the proteolysis was shown to be the cleavage of FP into the leader fragment and proinsulin. The subsequent splitting off of C-peptide from proinsulin results in the formation of ArgB31-ArgB32-insulin. The effect of temperature on the formation of de-ThrB30-insulin, a by-product, was also studied. The structure of FP was confirmed by the peptide mapping technique, and the leader fragment was shown to contain no N-terminal Met residue.     

Increased production of low molecular weight recombinant proteins in Escherichia coli.

A general method for obtaining high-level production of low molecular weight proteins in Escherichia coli is described. This method is based on the use of a novel Met-Xaa-protein construction which is formed by insertion of a single amino acid residue (preferably Arginine or Lysine) between the N-terminal methionine and the protein of interest. The utility of this method is illustrated by examples for achieving high-level production of human insulin-like growth factor-1, human proinsulin, and their analogs. Furthermore, highly produced insulin-like growth factor-1 derivatives and human proinsulin analogs are converted to their natural sequences by removal of dipeptides with cathepsin C.     

A single amino acid determinant of the membrane localization of lipoproteins in E. coli

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.     

用遗传工程的方法构建一个分泌型高表达的枯草杆菌碱性蛋白酶E(Subtilisin E)的枯草杆菌质粒-宿主系统

将克隆了枯草杆菌蛋白酶E基因aprE的枯草杆菌质粒pPZW101和一个组成型強启动子Psk连接构建成质粒pPZW102,转入碱性和中性蛋白酶缺失枯草杆菌DB104并进行表达。此质粒-宿主系统具有Subtilisin E高表达和外分泌的特点。