Characterization of the precursor of Serratia marcescens serine protease and COOH-terminal processing of the precursor during its excretion through the outer membrane of Escherichia coli.

The Serratia marcescens serine protease, which is directed by the gene encoding a precursor composed of a typical NH2-terminal signal sequence, a mature enzyme domain, and a large COOH-terminal domain, was excreted through the outer membrane of Escherichia coli. The precursor, with the expected molecular size (110 kilodaltons), was detected in an insoluble form in the periplasmic space of E. coli cells after induction with isopropyl-beta-D-thiogalactopyranoside of the expression of the gene under the control of the tac promoter. Upon membrane fractionation of the disrupted cells by sucrose density gradient centrifugation, the precursor was recovered from a fraction slightly heavier than the outer membrane fraction but not from the inner membrane fraction. Conversion of the precursor into the mature form, which was accompanied by its excretion into the medium, was observed even in the absence of de novo protein synthesis caused by the addition of chloramphenicol. The mutated gene product lacking all of the COOH-terminal domain was localized in the periplasmic space only and was not excreted into the medium. Additional mutant genes were generated by site-directed mutagenesis to test the role of some amino acids in the excretion of this protease in E. coli. The mutant protein with no protease activity because of the change of the catalytic residue Ser-341 to Thr was still excreted into the medium but with abnormal processing. Both self-processing and host-dependent processing of the precursor seem to be involved in the excretion of the mature enzyme. Replacement of the four Cys residues, two in the mature enzyme and two in the COOH-terminal domain, with Ser in different combinations caused a distinct or complete loss of excretion, suggesting that a certain conformation possibly formed via disulfide bonding was important for the excretion of the S. marcescens protease.     

Specific processing of the bacterial beta-lactamase precursor in Saccharomyces cerevisiae

Synthesis and processing of the bacterial enzyme beta-lactamase (E.C. 3.5. 2.6) were studied in Saccharomyces cerevisiae. The 2-micron DNA vector pADH040-2 containing the yeast ADH1 promoter fused to the bacterial gene was used in order to obtain enhanced synthesis of the bacterial protein in yeast transformants. Both precursor and mature beta-lactamase were shown to be present in yeast cells, the precursor being the major product. The mature enzyme was purified about 500-fold over crude extracts to apparent homogeneity and thus represents nearly 0.2% of the total yeast protein. No difference in specific activity and molecular weight could be observed when compared with the authentic beta-lactamase from Escherichia coli. Specificity of the processing of beta-lactamase in yeast cells was verified by partial amino acid sequence analysis demonstrating the removal of the signal peptide at the correct position.     

Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable

We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disulfide bonds as authentic hGH.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical.

Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals. Removal of the signals is carried out by specific, membrane-bound processing peptidases. In this report we show that the reaction specificities of these three peptidases are essentially identical. Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase. Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E. coli enzymes generates the correct mature amino terminus. Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase. No evidence of incorrect processing was obtained. Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.     

Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein

The expression of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of D. vulgaris DNA, containing the structural gene for cytochrome c3 (387 bp) and its promoter region. Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site [Ala(-1)----Asp(-1)]. Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E. coli TG2(pJ8) by SDS gel electrophoresis and Western blotting. Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction. No processing was observed in E. coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the E. coli product could be detected in E. coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome c3 synthesized in E. coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that E. coli is capable of synthesizing and exporting the cytochrome c3 polypeptide, but fails to insert the haems.     

大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为外膜蛋白前体

为进一步探讨大肠杆菌脑微血管内皮细胞侵袭基因ibeB的生物学特性 ,将ibeB基因克隆到pET2 8a(+)载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,经IPTG诱导后 ,通过Ni2 + NTA树脂提纯IbeB蛋白 .SDS PAGE确定纯化蛋白的分子量 ;应用无蛋白酶的体外转录和翻译系统进一步鉴定ibeB基因表达蛋白的分子量 ;通过 [3 5S]Met标记的体内T7表达体系并结合膜蛋白分离技术定位IbeB蛋白在细菌中的亚细胞分布 ;利用细菌侵袭实验分析IbeB蛋白抗体对E .coliK1侵袭人脑微血管内皮细胞的封闭作用 .结果发现 ,ibeB基因的重组蛋白表达纯化产物呈现出 5 0kD和 34kD两种分子量大小 ,5 0kD存在于表达细菌的可溶性部分 ,而 34kD则存在于包涵体中 ;体外翻译实验也显示出较弱的 5 0kD和较浓的 34kD两个蛋白带 ;体内T7表达体系实验显示 34kD的IbeB成熟蛋白定位于E .coli的外膜 ;抗 34kDIbeB蛋白抗体能封闭E .coli对人脑微血管内皮细胞的侵袭 .这些结果提示 ,大肠杆菌脑微血管内皮细胞侵袭基因ibeB的编码产物为 5 0kD的外膜蛋白前体 ,该前体可通过分子内剪接形成成熟的 34kDIbeB蛋白     

Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing

The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene. In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus. In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues. The pro-OmpA protein is normally processed partially co- and partially posttranslationally. Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational. Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane. The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis. Hence, it does not matter which mode of processing, or rather translocation, is used. The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor. In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues. This peptide was also rapidly removed. Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.     

Export defect adjacent to the processing site of staphylococcal nuclease is suppressed by a prlA mutation.

Plasmids have been constructed in which the Escherichia coli alkaline phosphatase promoter and signal sequence have been fused to the staphylococcal nuclease gene to promote the high-level expression and secretion of this gene product in E. coli. We determined that the first amino acid residue after the signal sequence can determine whether this protein was processed and exported to the periplasmic space. Fractionation and protease accessibility studies were used to show that the export-defective, nuclease precursor is internal to the cytoplasmic membrane barrier of the cell. Furthermore, this export defect was suppressed in a strain containing a prlA mutation. These findings are novel in that this region of the polypeptide chain has been implicated in processing but not export and that prlA mutations have not been previously known to suppress such defects.     

Expression of spinach plastocyanin in E. coli

An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence of Pseudomonas aeruginosa azurin and the mature sequence of spinach plastocyanin. The precursor is efficiently translocated to the periplasmic space and correctly processed to mature plastocyanin. No detectable amount of plastocyanin was present in the cytoplasmic or in the membrane fraction. A large scale preparation of the recombinant plastocyanin in a 20 litre fermentor yielded approximately 30 mg of pure plastocyanin. The recombinant protein obtained from E. coli shows CD, EPR and optical properties identical to plastocyanin isolated from spinach.     

Immediate entrance to the export pathway after synthesis as a requirement for export of the sak gene product in Escherichia coli.

Export through the cytoplasmic membrane and processing of the sak product in Escherichia coli cells were investigated with E. coli strains carrying pTS301, which produce large amounts of staphylokinase at 42 degrees C. High-level synthesis of the sak product caused transient accumulation not only of the staphylokinase precursor (pSAK) but also of the maltose-binding protein and outer membrane protein A precursors. Thus it was concluded that the sak product shares the export pathway with E. coli secreted proteins at least at a certain step. During high-level synthesis of the sak product, a significant amount of the newly synthesized pSAK remained unprocessed after a chase period, possibly causing the observed accumulation of pSAK. Accumulating pSAK did not mature for a long period, whereas the newly synthesized sak product was exclusively detected in the mature form. These results suggest that it is necessary for the sak product to enter the export pathway during or immediately after synthesis to be exported and processed normally.     

Defective Escherichia coli signal peptides function in yeast

To investigate structural characteristics important for eukaryotic signal peptide function in vivo, a hybrid gene with interchangeable signal peptides was cloned into yeast. The hybrid gene encoded nine residues from the amino terminus of the major Escherichia coli lipoprotein, attached to the amino terminus of the entire mature E. coli beta-lactamase sequence. To this sequence were attached sequences encoding the nonmutant E. coli lipoprotein signal peptide, or lipoprotein signal peptide mutants lacking an amino-terminal cationic charge, with shortened hydrophobic core, with altered potential helicity, or with an altered signal-peptide cleavage site. These signal-peptide mutants exhibited altered processing and secretion in E. coli. Using the GAL10 promoter, production of all hybrid proteins was induced to constitute 4-5% of the total yeast protein. Hybrid proteins with mutant signal peptides that show altered processing and secretion in E. coli, were processed and translocated to a similar degree as the non-mutant hybrid protein in yeast (approximately 36% of the total hybrid protein). Both non-mutant and mutant signal peptides appeared to be removed at the same unique site between cysteine 21 and serine 22, one residue from the E. coli signal peptidase II processing site. The mature lipo-beta-lactamase was translocated across the cytoplasmic membrane into the yeast periplasm. Thus the protein secretion apparatus in yeast recognizes the lipoprotein signal sequence in vivo but displays a specificity towards altered signal sequences which differs from that of E. coli.     

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.     

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.     

High level expression of a protein precursor for functional studies

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial preapo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.     

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.     

Cloning and characterization of the secY gene from the cyanobacterium Synechococcus PCC7942.

The secY gene product is an essential component of the Escherichia coli cytoplasmic membrane, which mediates the protein translocation across the membrane. We found a gene homologous to secY in the genome of the cyanobacterium Synechococcus PCC7942. The deduced amino acid sequence, 439 amino acids long, shows 43% homology with that of the E. coli secY. The hydrophobic profile suggests that the Synechococcus SecY protein is an integral membrane protein containing ten membrane-spanning segments, which are closely related to the E. coli counterpart. The SecY protein may participate in the protein translocation across the cytoplasmic or thylakoid membrane in Synechococcus PCC7942.     

Expression of rat alpha-fetoprotein cDNA in Escherichia coli and in yeast

Rat alpha-fetoprotein (AFP) cDNA spanning the complete coding region was cloned and expressed in Escherichia coli as well as in yeast, Saccharomyces cerevisiae. The recombinant AFPs (rAFPs) were purified and characterized. The molecular weights determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were 65,000 for E. coli rAFP and 69,000 for yeast rAFP. Amino acid and N-terminal sequence analyses indicated that yeast cells produced mature AFP by processing the signal peptide properly but E. coli rAFP lacked the N-terminal 53 amino acid residues of preAFP. The yeast rAFP was found to be indistinguishable from authentic AFP in the Ouchterlony immunodiffusion test, radioimmunoassay and estradiol-binding assay while E. coli rAFP was less reactive in these tests. These observations indicated that the rAFP expressed in yeast emulated the properties of authentic AFP.