Escherichia coli skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments

Expression of single-chain antibody fragments (scAb)in the periplasm of Escherichia coli often results in low soluble product yield and cell lysis. We have increased scAb solubility and prevented cell culture lysis by coexpressing the E. coli Skp chaperone gene. A mutant Skp cistron was linked to a bacteriophage T7 gene 10 translational initiation region and placed either downstream of a scAb gene within an isopropyl beta-d-thiogalactopyranoside-inducible expression cassette or on a separate colE1-compatible arabinose-inducible vector. Increases in scAb solubility reflected the amount of coexpressed Skp. A bacteriophage display vector that was also engineered to coexpress Skp permitted display of a virtually undisplayable scAb and should prove useful in expanding library sizes.     

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

Background  

The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression.     

An auto-inducible Escherichia coli lysis system controlled by magnesium

The Escherichia coli (E. coli) prokaryotic expression system is widely used in the field of biology. The currently adopted processes for inducing cell wall rupture, in order to release the target protein, are complex and cumbersome. We developed an auto-inducible E. coli lysis system that is regulated by exogenous magnesium ion (Mg²⁺) concentration. This system is composed of a strictly Mg²⁺-regulated promoter Pmgt from the mgtB gene of Salmonella typhimurium, and the lysis genes from λ bacteriophage. Both the wild type and Sam7-mutant lysis genes were inducibly expressed in E. coli under Mg²⁺-depleted conditions. The former caused a rapid lysis, while the latter induced very mild lysis of the host strains. However, rapid lysis was observed when the latter was resuspended in Tris–EDTA buffer. Finally, the inducible lysis cassette containing wild type lysis gene was introduced into an expression plasmid expressing GFP gene and efficient lysis of the host E. coli strain and subsequent release of the target protein was achieved in Mg²⁺-depleted conditions. Collectively, the current study indicates that this novel inducible lysis system could have attractive applications in the field of protein expression and provides new insights for the development of bacterium-based vaccines.     

A bifunctional vector system for controlled expression and subsequent release of the cloned gene product by øX174 lysis protein-E

Summary A new bifunctional Escherichia coli cloning vector, pSB50, is presented. The plasmid allows controlled expression of a gene of interest under control of the lac promoter and the subsequent release of the cloned product by the use of bacteriophage øX174 lysis protein-E, the gene of which is under control of the phage Lambda pL promoter. To ensure optimal repression of the Lambda pL promoter and the lac promoter in plasmid pSB50, E. coli strain UB89-1 was constructed which carries a chromosomal copy of the lambda cl857 repressor allele and the lacl ^q1 allele, respectively. Here, we employ the E-based lysis system to release human prourokinase to the culture medium. Offprint requests to: U. Bläsi     

Heterologous expression of functionally active enterolysin A, class III bacteriocin from Enterococcus faecalis, in Escherichia coli

The heterologous expression of enterolysin A (EnlA), heat-labile class III bacteriocin from Enterococcus faecalis II/1 with anti-listerial activity, was studied in Escherichia coli. The PCR amplified products of enterolysin A structural gene, N-terminal part of EnlA with endopeptidase-like activity and C-terminal part of EnlA similar to a lysis gene of bacteriophage, were cloned in prelinearized pQE-30UA expression vector. The expression of EnlA structural gene led to the synthesis and secretion of functional-active His-tagged enterolysin A protein, which was purified to homogeneity using His-Select™ Cartridge and was shown to be fully active against the indicator strain. The expression of N-terminal or C-terminal part of EnlA and deletion of last 58 amino acids from C-terminal domain of EnlA led to the synthesis of biologically non-active proteins.     

High-level production of a single chain antibody against anthrax toxin in Escherichia coli by high cell density cultivation

Previously, we isolated the M18 scFv, which is an affinity matured antibody against the anthrax toxin PA, and observed that its single chain antibody (scAb) form (M18 scAb) exhibited superior stability compared to the scFv. Here, we report high cell density cultivations for preparative scale production of M18 scAb in a 3.5 L fermenter. Briefly, a pH–stat feeding strategy was employed in fed-batch cultivation, and four different cell densities (OD₆₀₀ of 40, 80, 120, and 150) were examined for the induction of scAb gene expression. Among the four cell densities investigated, lower cell densities (OD₆₀₀ of 40) showed higher post-induction cell growth and production yields (665 mg/L of scAb). Even though lower solubility (51%) of scAb was achieved at lower cell density (OD₆₀₀ of 40), monomeric scAb could be purified with high purity (>95%) using simple purification procedures. The purified scAb from high cell density cultures showed biological activity equivalent to that of scAb purified from shake flask cultivation.     

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter⁻¹) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.     

Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Complementation of Bacteriophage Induction and Recombination Defects in <Emphasis Type="Italic">Escherichia coli</Emphasis> RecA<Superscript>−</Superscript> Mutants by Expression of the Cloned T4 Bacteriophage <Emphasis Type="Italic">uvsX</Emphasis> Gene

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA⁻ mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present. Received: 11 February 2002 / Accepted: 12 April 2002     

Thymol-triggered lysis of Escherichia coli expressing Lactobacillus phage LL-H muramidase

Effective disruption of Escherichia coli cells is achieved by the intracellularly accumulated recombinant murein hydrolase (Lactobacillus bacteriophage LL-H muramidase) after the addition of 5 mM thymol. Thymol destroys the integrity and electric potential of the cytoplasmic membrane, and as a consequence the muramidase can access and hydrolyze the cell wall murein leading to cell lysis. Lysis occurred within 5 min after the addition of thymol and seemed to be efficient at high culture densities. This lysis method does not require cell harvesting or addition of other cell wall weakening substances or exogenous enzymes. As a cell disruption method, thymol-triggered lysis is as efficient as sonication in the presence of 1% Triton. Furthermore, thymol did not interfere with the purification steps of Mur by expanded bed adsorption chromatography (EBA), suggesting that the lysis method presented here is well suited for large-scale production and purification of intracellular proteins of E. coli. Received 21 April 1998/ Accepted in revised form 5 December 1998     

Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli

Expression of cloned gene E of bacteriophage PhiX174 induces lysis by formation of a transmembrane tunnel structure in the cell envelope of Escherichia coli. Ultrastructural studies of the location of the lysis tunnel indicate that it is preferentially located at the septum or at polar regions of the cell. Furthermore, the diameter and shape of individual tunnel structures vary greatly indicating that its structure is not rigid. Apparently, the contours of individual lysis tunnels are determined by enlarged meshes in the peptidoglycan net and the force produced at its orifice, by the outflow of cytoplasmic content. Once the tunnel is formed the driving force for the lysis process is the osmotic pressure difference between cytoplasm and medium. During the lysis process areas of the cytoplasmic membrane which are not tightly attached to the envelope are extended inward by the negative pressure produced during lysis. After cell lysis external medium can diffuse through the lysis tunnel filling the inner cell space of the still rigid bacterial ghosts.     

High-level expression of Clostridium thermocellum cellulase genes in Escherichia coli

Summary The Clostridium thermocellum cellulase genes celA and celC encoding endoglucanase A and C were subcloned in a temperature-regulated Escherichia coli expression vector containing the leftward promoterpl of bacteriophage lambda. The level of gene expression was controlled by thermal inactivation of the heat-sensitive lambda cI857 repressor. Under optimal conditions the recombinant endoglucanases A and C were expressed to a level of 10–15% of total cellular protein. Endoglucanase A was partially exported into the periplasmic space, whereas endoglucanase C was found sequestered within the cytoplasm. Overexpression of the celA gene resulted in decreased cell viability concomitant with the accumulation of endoglucanase A in the membrane fraction. In contrast, high-level synthesis of the celC gene product was well tolerated by the host cell. Overproduced endoglucanase C accumulated as a soluble enzyme without detectable formation of inactive inclusion bodies.     

KL2型鲍曼不动杆菌噬菌体解聚酶克隆及抗菌活性分析

【背景】噬菌体解聚酶是噬菌体在裂解细菌过程中产生的一种抗菌蛋白,关于鲍曼不动杆菌荚膜分型及常见型别噬菌体解聚酶的研究报道较少。【目的】以KL2型鲍曼不动杆菌为研究对象,从噬菌体IME-AB2中克隆解聚酶,在大肠杆菌中进行可溶性表达并研究其体外抗菌活性。【方法】应用二代测序及生物信息学方法鉴定鲍曼不动杆菌荚膜型,分析IME-AB2全基因组。应用分子克隆技术克隆ORF76假定的尾丝蛋白(Putative Tail Fiber)基因,构建重组表达载体pEASY-Blunt-E1-gp76,在大肠杆菌BL21(DE3)中诱导表达,通过Ni-NTA亲和层析纯化解聚酶,研究解聚酶体外抗菌活性。【结果】构建了pEASY-Blunt-E1-gp76解聚酶重组表达质粒,该重组质粒在大肠杆菌中得到可溶性表达;体外活性分析显示,该重组蛋白在体外能够对所有的KL2型鲍曼不动杆菌具有较好的抗菌活性,解聚酶联合人和狗的血清具有很好的杀菌活性。【结论】鉴定解聚酶并提高其抗菌谱具有重要意义,也是噬菌体及解聚酶用于治疗耐药菌研究领域急需解决的重要问题之一。     

Chromosomal duplications and cointegrates generated by the bacteriophage lamdba Red system in Escherichia coli K-12

Background  

An Escherichia coli strain in which RecBCD has been genetically replaced by the bacteriophage λ Red system engages in efficient recombination between its chromosome and linear double-stranded DNA species sharing sequences with the chromosome. Previous studies of this experimental system have focused on a gene replacement-type event, in which a 3.5 kbp dsDNA consisting of the cat gene and flanking lac operon sequences recombines with the E. coli chromosome to generate a chloramphenicol-resistant Lac- recombinant. The dsDNA was delivered into the cell as part of the chromosome of a non-replicating λ vector, from which it was released by the action of a restriction endonuclease in the infected cell. This study characterizes the genetic requirements and outcomes of a variety of additional Red-promoted homologous recombination events producing Lac+ recombinants.     

Improved expression characteristics of single-chain Fv fragments when fused downstream of the Escherichia coli maltose-binding protein or upstream of a single immunoglobulin-constant domain

The expression of single-chain Fv fragments (scFv) targeted to the periplasm of Escherichia coli often results in very low yields of soluble protein frequently accompanied by host cell growth arrest and sometimes lysis. Single-chain antibody fragments (scAb) are scFv with a human kappa light chain constant (HuCkappa) domain attached C-terminally and share similar problems of expression. By fusing the E. coli maltose-binding protein (mbp) gene either 3' or 5' to a scAb specific for the herbicide atrazine, a reduction in growth arrest was observed that was dependent on the order of gene fusion. The scAb-mbp fusion delayed the onset of growth arrest following induction while the mbp-scAb fusion appeared to ablate growth arrest completely. Cell fractionation revealed barely detectable levels of scAb-mbp in the periplasm while mbp-scAb was detected at equivalent levels as scAb in the periplasmic compartment, indicating that periplasmic scAb solubility is unrelated to propensity to cause growth arrest. IMAC purification of scAb and mbp-scAb proteins followed by liquid competition ELISA revealed the IC(50) for atrazine to be approximately 1 nM for both proteins demonstrating that 5'-mbp fusion does not alter antigen binding. The equivalent scFv and mbp-scFv vectors expressed far less material in both periplasmic and insoluble fractions indicating that the HuCkappa domain can have a positive effect on scFv expression when expressed either alone or as a mbp fusion. The ablation of growth arrest by a 5'-mbp fusion and enhancement of expression by a 3'-HuCkappa domain fusion were extended to a second scFv specific for the herbicide diuron. Therefore, by expressing scFv as tripartite fusions (mbp-scFv-HuCkappa) enhanced levels of soluble periplasmic expression can be achieved without causing growth arrest of the host cell, realizing the potential for constitutive expression of hapten-binding scFv in the E. coli periplasm.     

Autotransporter-Based Antigen Display in Bacterial Ghosts

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.     

Cloning and characterization of the gene, speC, for pyrogenic exotoxin type C from Streptococcus pyogenes

Summary The structural gene of streptococcal pyrogenic exotoxin type C (SPE C) was cloned from the chromosome of Streptococcus pyogenes strain T18P into Escherichia coli using pBR328 as the vector plasmid. Subcloning enabled the localization of the gene (speC) to a 1.7 kb fragment. Partially purified E. coli-derived SPE C and purified streptococcal-derived SPE C, were shown to have the same molecular weight (23 800) and biological activities. A DNA probe, prepared from cloned speC, cross-hybridized with the structural genes of SPE A and SPE B indicating relatedness at the nucleotide level. The speC-derived probe also hybridized to a fragment of CS112 bacteriophage DNA containing the phage attachment site.     

Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis

Summary The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal -endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. colicells by disrupting their chromosome replicating apparatus.     

Additional Volatile Compounds Produced by Pyrolysis of Sulfur-containing Amino Acids

Bacillus circulans WL-12, a yeast and fungal cell wall lytic bacterium, secretes a variety of polysaccharide degrading enzymes into the culture medium. When β-1,3-glucanase was induced with pachyman, a β-1,3-glucose polymer obtained from the tree fungus Poria cocus Wolf, six distinct active molecules of the enzyme with different molecular weights were detected in the culture supernatant of this bacterium. Molecular cloning of one of the β,3-gIucanase genes into E. coli was achieved by transforming E. coli HB101 cells with recombinant plasmids composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pUC 19. A recombinant plasmid containing 4.4 kb of inserted DNA in the Pst I site of pUC 19, designated as pNT003, conferred the ability to degrade pachyman on E. coli cells. The presence of pNT003 was harmful for E. coli cells and caused cell lysis, especially at higher temperatures of cultivation. β,3-Glucanase activity detected in E. coli was mainly recovered in the periplasmic fraction when cell lysis did not occur. SDS-PAGE analysis revealed that the periplasmic fraction contained four active molecules of β-1,3-glucanase which corresponded to four of the six active molecules produced by B. circulans WL-12.     

Enhancing Functional Expression of Heterologous <Emphasis Type="Italic">Burkholderia</Emphasis> Lipase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm of E. coli was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in enhancing the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm of E. coli formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip–HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.