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1.
Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification. However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression. The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community. 相似文献
2.
Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA. 相似文献
3.
Identification of individual amino acids required for secretion within the haemolysin (HlyA) C-terminal targeting region 总被引:8,自引:0,他引:8
The release of haemolysin from Escherichia coli involves direct secretion across both the inner and outer membranes. Secretion of HlyA is dependent upon a specific membrane export complex composed of HlyB, -D and possibly TolC. HlyA is targeted to the medium via the membrane translocation complex, by a novel C-terminal secretion signal. Previous studies involving deletion and fusion analyses have given contradictory results for the minimal length (20-60 residues) of this HlyA signal region and little is known of the nature of the specific residues and structural features required for function. In this study we have analysed, quantitatively, the effect upon secretion of many point mutations introduced into the HlyA C-terminus. The results indicate the presence of a minimal secretion signal domain whose proximal boundary extends to at least residue -46 and which contains at least four individual residues essential for maximal secretion levels. We propose that such residues act co-operatively, forming multiple contact points with the translocator proteins, with the 'best fit' promoting maximal levels of secretion. 相似文献
4.
【目的】构建一株具备外分泌蛋白功能的工程菌,解决杀虫毒素无法由胞内分泌至胞外,无法直接作用于虫体等问题,为松墨天牛防治提供新思路。【方法】本研究先测定从松墨天牛肠道及其生境中分离出的嗜虫耶尔森氏菌(CSLH88)的生长特性及抗性,进而对其进行分子改造。构建HlyA (pGHKW2)以及HasA (pGHKW4)外分泌表达载体,利用电穿孔法将其转入CSLH88菌株,获得能够表达绿色荧光蛋白的工程菌。利用稀释涂板及荧光体式镜检测技术对两个质粒进行遗传稳定性检测,并采用SDS-PAGE及Western blotting技术验证蛋白外分泌功能。【结果】CSLH88菌株培养2–4 h能够进入对数生长期,并对卡那霉素(Kan)敏感。成功构建了含有Kan抗性基因的pGHKW2(GenBank:MK562405)和pGHKW4(GenBank:MK562404)两个外分泌表达载体的CSLH88工程菌株。其中,发现pGHKW4质粒更加适合在嗜虫耶尔森氏菌中稳定遗传。SDS-PAGE及Western blotting检测结果表明HlyA系统无法在CSLH88菌株中将目的蛋白分泌到胞外,而HasA系统则可以有效地发挥外分泌表达功能。【结论】通过对HlyA及HasA两个外分泌表达系统进行研究,从中筛选出HasA型血红素转运系统作为CSLH88菌株的外分泌表达系统,为后续外分泌杀虫毒素蛋白菌株构建以及CSLH88菌株的致病性研究奠定基础。 相似文献
5.
Ralf Schülein Ivaylo Gentschev Stefan Schlör Roy Gross Werner Goebel 《Molecular & general genetics : MGG》1994,245(2):203-211
Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus. 相似文献
6.
Active and inactive forms of hemolysin (HlyA) from Escherichia coli 总被引:11,自引:0,他引:11
The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase. 相似文献
7.
T. Jarchau T. Chakraborty F. Garcia W. Goebel 《Molecular genetics and genomics : MGG》1994,245(1):53-60
Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5′ end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA. 相似文献
8.
The mechanism of secretion of hemolysin and other polypeptides from Gram-negative bacteria 总被引:5,自引:0,他引:5
I. Barry Holland Mark A. Blight Brendan Kenny 《Journal of bioenergetics and biomembranes》1990,22(3):473-491
In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of theE. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD. 相似文献
9.
Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins. 总被引:10,自引:5,他引:5
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R L Oropeza-Wekerle E Müller P Kern R Meyermann W Goebel 《Journal of bacteriology》1989,171(5):2783-2788
Extra- and intracellular Escherichia coli hemolysin expressed by two cloned hly determinants, both under the control of the activator element hlyR, were analyzed. One determinant carried all four hly genes (hlyC, hlyA, hlyB, and hlyD), whereas the other carried only the two genes (hlyC and hlyA) required for synthesis of active hemolysin but not those essential for its secretion. It was shown that the total amounts of HlyA protein and of hemolytic activity are similar in both cases in logarithmically growing cultures. The E. coli strain carrying the complete hly determinant released most hemolysin into the media and accumulated very little HlyA intracellularly. The active extracellular hemolysin (HlyA*) was inactivated in the stationary phase without degradation of the HlyA protein. In contrast, the hemolysin which accumulated intracellularly in the E. coli strain carrying hlyA and hlyC only was proteolytically degraded at the end of the logarithmic growth phase. Immunogold labeling indicates that active intracellular HlyA bound preferentially to the inner membrane, whereas that part of the extracellular HlyA which remained cell-bound was located exclusively at the cell surface. It was shown by fluorescence-activated cell sorter analysis that active extra- and intracellular HlyA* bound with similar efficiency to erythrocytes, whereas hemolytically inactive HlyA protein did not bind to these target cells. 相似文献
10.
HlyA is a toxin secreted by uropathogenic Escherichia coli strains. HlyA belongs to the repeats in the toxin protein family and needs (i) a posttranslational, fatty acylation at two internal lysines by the acyltransferase HlyC and (ii) extracellular ion binding to achieve its active conformation. Both processes are not fully understood and experiments are often limited due to the low amounts of protein available. Here, we present an optimized purification protocol for the proteins involved in HlyA activation as well as a quick and nonradioactive assay for in vitro HlyA acylation. These may simplify future experiments, e.g., activity scanning and characterization of HlyA or HlyC mutants as demonstrated with single and double HlyA lysine mutants. 相似文献
11.
Pseudomonas aeruginosa releases several extracellular proteins which are secreted via two independent secretion pathways. Alkaline protease (AprA) is released by its own specific secretion machinery which is an ABC-transporter. Despite sequence similarities between components of ABC-transporters in different bacteria, each transporter is dedicated to the secretion of a particular protein or a family of closely related proteins. Heterologous complementation between ABC-transporters for unrelated polypeptides can occur, but only at a very low level. We show that the 50 C-terminal amino acids of AprA constitute an autonomous secretion signal. By heterologous complementation experiments between the unrelated a-haemolysin (HlyA) and Apr secretion systems we demonstrated that it is only the recognition of the secretion signal by the trans-locator which confers specificity to the secretion process. Secretion was size-dependent. However inclusion of glycine-rich repeats from HlyA in AprA seems to overcome the size limitation exerted by the Apr secretion apparatus such that the machinery secreted a hybrid protein 20kDa larger than the normal maximal size. 相似文献
12.
Niju Narayanan Manal Khan C. Perry Chou 《Journal of industrial microbiology & biotechnology》2010,37(4):349-361
Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli has been technically problematic due to protein misfolding, ineffective disulfide bond formation, and protein instability
associated with intracellular proteolysis. To overcome these problems, an alternative approach was explored in this study
by extracellular secretion of PalB via two Sec-independent secretion systems, i.e., the α-hemolysin (type I) and the modified
flagellar (type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior
of the cell. Both shaker flask and bioreactor cultivations were performed to characterize the developed PalB expression/secretion
systems. Bioactive PalB was expressed and secreted extracellularly either as a HlyA fusion (i.e., PalB-HlyA via type I system)
or an intact protein (via type III system). However, the secretion intermediates in the intracellular fraction of culture
samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development
of PalB activity. Also importantly, the secretion strategy appeared to have a minimum impact on cell physiology. PalB secretion
via the type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion
via the type III system was slow with lower specific PalB activities but effective cell growth. 相似文献
13.
Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain. 相似文献
14.
The repeat domain of Escherichia coli haemolysin (HlyA) is responsible for its Ca2+-dependent binding to erythrocytes 总被引:29,自引:0,他引:29
Albrecht Ludwig Thomas Jarchau Roland Benz Werner Goebel 《Molecular & general genetics : MGG》1988,214(3):553-561
Summary The haemolysin protein (HlyA) of Escherichia coli contains 11 tandemly repeated sequences consisting of 9 amino acids each between amino acids 739 and 849 of HlyA. We removed, by oligonucleotide-directed mutagenesis, different single repeats and combinations of several repeats. The resulting mutant proteins were perfectly stable in E. coli and were secreted with the same efficiency as the wild-type HlyA. HlyA proteins which had lost a single repeat only were still haemolytically active (in the presence of HlyC) but required elevated levels of Ca2+ for activity, as compared to the wild-type haemolysin. Removal of three or more repeats led to the complete loss of the haemolytic activity even in the presence of high Ca2+ concentrations. The mutant haemolysins were unable to compete with the wild-type haemolysin for binding to erythrocytes at low Ca2+ concentrations but could still generate ion-permeable channels in artificial lipid bilayer membranes formed of plant asolectin, even in the complete absence of Ca2+. These data indicate that the repeat domain of haemolysin is responsible for Ca2+-dependent binding of haemolysin to the erythrocyte membrane. A model for the possible functional role of Ca2+ in haemolysis is presented. 相似文献
15.
Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation. 相似文献
16.
McLeod J O'Callaghan PM Pybus LP Wilkinson SJ Root T Racher AJ James DC 《Biotechnology and bioengineering》2011,108(9):2193-2204
17.
Bakkes PJ Jenewein S Smits SH Holland IB Schmitt L 《The Journal of biological chemistry》2010,285(52):40573-40580
Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function. 相似文献
18.
Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC.
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Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of residue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level. Complete inactivation of HlyC was caused by a double mutation, replacement of Gly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dimensional gel electrophoresis revealed the production and simultaneous secretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites. 相似文献
19.
Hideo Nakano Yasushi Kawakami Hajime Nishimura 《Applied microbiology and biotechnology》1992,37(6):765-771
Summary Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli -haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a -sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.
Correspondence to: H. Nakano 相似文献
20.
【背景】重组酿酒酵母可用于生产多种药用蛋白和工业酶等外源蛋白,但蛋白分泌水平低是限制其异源蛋白高效生产的重要因素。异源蛋白表达和分泌过程可能会对宿主细胞产生多种胁迫,因此,研究胁迫响应相关基因对重组酵母异源蛋白生产的影响具有重要意义。Mhf1p是MHF组蛋白折叠复合体的组分之一,与DNA损伤修复及维持基因组稳定性有关,但其对异源蛋白生产的作用尚不清楚。【目的】研究MHF1过表达对重组酿酒酵母蛋白生产的影响。【方法】在分泌表达纤维素酶的重组酿酒酵母菌株中利用基于CRISPR-Cas9的基因组编辑技术整合过表达MHF1,分析其对产酶的影响,并探讨影响产酶的分子机理。【结果】与出发菌株相比,过表达MHF1菌株的外切纤维素酶CBH酶活性提高了38%。对过表达MHF1的CBH生产菌株中蛋白合成和分泌途径相关基因转录水平进行检测,发现与对照菌株相比,CBH1基因和与分泌相关的SEC22、ERV29等基因在不同时间点呈现不同程度显著上调。【结论】MHF1过表达可促进酿酒酵母异源外切纤维素酶的生产,并影响外源酶基因和分泌途径基因的表达,可能通过对多基因的协同表达影响促进产酶。 相似文献