In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA.

The two-domain, 325 residue outer membrane protein OmpA of Escherichia coli is a well-established model for the study of membrane assembly. The N-terminal domain, consisting of approximately 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta-barrel consisting of eight antiparallel transmembrane beta-strands. A set of 16 gene variants carrying deletions in the membrane-embedded domain of OmpA was constructed. When pairs of these mutant genes were co-expressed in E.coli, it was found that a functional OmpA protein could be assembled efficiently from two complementary protein fragments. Assembly was found when the polypeptide chain was split at the second or third periplasmic turn. All four protein termini were located in the periplasmic space. Interestingly, duplication of transmembrane strands five and six led to a variant with an unusual topology: the N-terminus of one fragment and the C-terminus of the other fragment were exposed at the cell surface. This is the first demonstration of correct membrane assembly of split beta-structured membrane proteins. These findings are important for a better understanding of their folding/assembly pathway and may have implications for the development of artificial outer membrane proteins and for the cell surface display of heterologous peptides or proteins.     

A structurally informed autotransporter platform for efficient heterologous protein secretion and display

ABSTRACT: BACKGROUND: The self-sufficient Autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a beta-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. RESULTS: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a beta-helical core structure (beta-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated beta-stem appeared unstable after translocation, demonstrating the importance of an intact beta-stem. By interrupting the cleavage site between passenger and beta-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. CONCLUSIONS: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.     

Functional cell surface display and controlled secretion of diverse Agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10(4) molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.     

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.     

The autodisplay story, from discovery to biotechnical and biomedical applications.

Among the pathways used by gram-negative bacteria for protein secretion, the autotransporter pathway represents a solution of impressive simplicity. Proteins are transported, independent of their nature as recombinant or native passengers, as long as the coding nucleotide sequence is inserted in frame between those of an N-terminal signal peptide and a C-terminal domain, referred to as the beta-barrel of the outer membrane translocation unit. The immunoglobulin A1 (IgA1) protease from Neisseria gonorrhoeae was the first identified member of the autotransporter family of secreted proteins. The IgA1 protease was employed in initial experiments investigating autotransporter-mediated surface display of recombinant proteins and to investigate structural and functional requirements. Various other autotransporter proteins have since been described, and the autodisplay system was developed on the basis of the natural Escherichia coli autotransporter protein AIDA-I (adhesin involved in diffuse adherence). Autodisplay has been used for the surface display of random peptide libraries to successfully screen for novel enzyme inhibitors. The autodisplay system was also used for the surface display of functional enzymes, including esterases, oxidoreductases, and electron transfer proteins. Whole E. coli cells displaying enzymes have been utilized to efficiently synthesize industrially important rare organic compounds with specific chirality. Autodisplay of epitopes on the surface of attenuated Salmonella carriers has also provided a novel way to induce immune protection after oral vaccination. This review summarizes the structural and functional features of the autodisplay system, illustrating its discovery and most recent applications. Autodisplay facilitates the export of more than 100,000 recombinant molecules per single cell and permits the oligomerization of subunits on the cell surface as well as the incorporation of inorganic prosthetic groups after transport of apoproteins onto the bacterial surface without disturbing bacterial integrity or viability. We discuss future biotechnical and biomedical applications in the light of these achievements.     

Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli

The surface display of heterologous proteins on live Escherichia coli using anchoring motifs from outer membranes proteins has impacted on many areas of biochemistry, molecular biology and biotechnology. The Anaplasma marginale major surface protein 1a (MSP1a) contains N-terminal surface-exposed repeated peptides (28-289 amino acids) that are involved in pathogen interaction with host cell receptors and is surface-displayed when the recombinant protein is expressed in E. coli. Therefore, it was predicted that MSP1a would surface display on E. coli peptides inserted in the N-terminal repeats region of the protein. The Rhipicephalus (Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that a recombinant protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region is displayed on the E. coli surface and is recognized by anti-BM86 and anti-MSP1a antibodies. This system provides a novel approach to the surface display of heterologous antigenic proteins on live E. coli and suggests the possibility to use the recombinant bacteria for immunization studies against cattle tick infestations.     

Bacterial proteins can be processed by macrophages in a transporter associated with antigen processing-independent, cysteine protease-dependent manner for presentation by MHC class I molecules

MHC class I molecules present peptides derived primarily from endogenously synthesized proteins on the cell surface as ligands for CD8+ T cells. However, CD8+ T cell responses to extracellular bacteria, virus-infected, or tumor cells can also be elicited because certain professional APC can generate peptide/MHC class I (MHC-I) complexes from exogenous sources. Whether the peptide/MHC-I complexes are generated because the exogenous proteins enter the classical cytosolic, TAP-dependent MHC-I processing pathway or an alternate pathway is controversial. Here we analyze the generation of peptide/MHC-I complexes from recombinant Escherichia coli as an exogenous Ag source that could be delivered to the phagosomes or directly into the cytosol. We show that peritoneal and bone marrow macrophages generate peptide/MHC-I complexes by the classical as well as an alternate, but relatively less efficient, TAP-independent pathway. Using a novel method to detect proteolytic intermediates we show that the generation of the optimal MHC-I binding peptide in the alternate pathway requires cysteine as well as other protease(s). This alternate TAP-independent pathway also operates in vivo and provides a potential mechanism for eliciting CD8+ T cell responses to exogenous Ags.     

Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins

We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.     

Requirements for chemotaxis protein localization in Rhodobacter sphaeroides

Many proteins have recently been shown to localize to different regions of the bacterial cell. This is most striking in the case of the Escherichia coli chemotaxis pathway in which the components localize at the cell poles. Rhodobacter sphaeroides has a more complex chemotaxis system with two complete pathways, each localizing to different positions, one pathway at the pole and one at a discrete cluster within the cytoplasm of the bacterium. Using genomic replacement of the wild-type chemotaxis genes in R. sphaeroides with their corresponding fluorescent protein fusions in conjunction with in frame deletions of other chemotaxis genes, we have investigated which proteins are required for the formation of the polar and cytoplasmic chemotaxis protein clusters. As in E. coli, the polarly targeted CheA and CheW homologues are required for the formation of the polar cluster. However, the formation of the cytoplasmic cluster requires the cytoplasmic chemoreceptors and CheW but not the CheAs. Interestingly, even when deletion of a component resulted in the chemotaxis proteins of one pathway becoming delocalized and diffuse in the cytoplasm, in no case were any chemotaxis proteins seen to localize to the other signalling cluster.     

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.     

A class of Escherichia coli proteins controlled by the hflA locus

The HflA protein of Escherichia coli is critical for the choice of the lytic or lysogenic pathway by bacteriophage lambda. To investigate whether HflA plays a regulatory role in E. coli, we used two-dimensional gel electrophoresis to compare the distribution of E. coli proteins in hflA+ and hflA- cells. We found at least 13 proteins that are present in hflA- strains, but absent or very low in hflA+ strains. This observation indicates that HflA might be involved in regulation of a large class of E. coli proteins. Because of prior work implicating the Crp/cAMP system in Hfl-mediated regulation of lambda, we also studied the distribution of E. coli proteins in cya- strains unable to synthesize cAMP. Some of the same proteins found in hflA- are elicited by the addition of cAMP to cya- cells.     

Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein

We investigated the ability of the N-terminal domain of InaK, an ice nucleation protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of foreign proteins on the Escherichia coli cell surface. Total expression level and surface display efficiency of green fluorescent protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC). We report that the InaK-N/GFP fusion protein showed a similar cell surface display efficiency ( approximately 50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif. Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density. InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif.     

SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli

Antibody phage display is a key technology for the generation of recombinant (human) antibodies for research, diagnostics and therapy. Most antibody fragments can only be folded correctly in the oxidizing environment of the periplasm of Escherichia coli. A multitude of leader peptides has been used for secretion of antibody::pIII fusion proteins into the periplasm, but a systematic study of their impact on the performance of antibody phage display systems has not been reported so far. In this work we have analysed the influence of various leader peptides on antibody phage display efficiency and production yields of soluble antibody fragments. Four leader peptides using the Sec pathway (PelB, OmpA, PhoA and pIII) and three using the SRP pathway (DsbA, TorT and TolB) were compared. Both pathways are compatible with antibody phage display and the production of soluble antibody fragments. The applicability of the SRP pathway to antibody phage display and the production of functional scFvs is shown here for the first time.     

Diverse paths to midcell: assembly of the bacterial cell division machinery

At the heart of bacterial cell division is a dynamic ring-like structure of polymers of the tubulin homologue FtsZ. This ring forms a scaffold for assembly of at least ten additional proteins at midcell, the majority of which are likely to be involved in remodeling the peptidoglycan cell wall at the division site. Together with FtsZ, these proteins are thought to form a cell division complex, or divisome. In Escherichia coli, the components of the divisome are recruited to midcell according to a strikingly linear hierarchy that predicts a step-wise assembly pathway. However, recent studies have revealed unexpected complexity in the assembly steps, indicating that the apparent linearity does not necessarily reflect a temporal order. The signals used to recruit cell division proteins to midcell are diverse and include regulated self-assembly, protein-protein interactions, and the recognition of specific septal peptidoglycan substrates. There is also evidence for a complex web of interactions among these proteins and at least one distinct subcomplex of cell division proteins has been defined, which is conserved among E. coli, Bacillus subtilis and Streptococcus pneumoniae.     

Cell surface display of salmobin, a thrombin-like enzyme from Agkistrodon halys venom on Escherichia coli using ice nucleation protein

Cell surface display on Escherichia coli using ice nucleation protein was performed in order to develop a new expression system for recombinant eukaryotic proteins. Salmobin, the thrombin-like enzyme obtained from Korean snake (Agkistrodon halys) venom was displayed on the surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. The thrombin cleavage site was inserted between salmobin and INP. The presence of salmobin on the bacterial cell surface was verified by SDS-PAGE, Western blotting, whole cell ELISA, and immunofluorescence microscopy. After thrombin cleavage the thrombin-like enzyme activity of recombinant salmobin was tested and verified. We concluded that INP-based cell surface display can be used as a novel expression system for eukaryotic proteins.     

Bacterial display in combinatorial protein engineering

Technologies for display of recombinant protein libraries are today essential tools in many research-intensive fields, such as in the drug discovery processes of biopharmaceutical development. Phage display is still the most widely used method, but alternative systems are available and are becoming increasingly popular. The most rapidly expanding of the alternative systems are the cell display-based technologies, offering innovative strategies for selection and characterization of affinity proteins. Most investigations have focused on eukaryotic yeast for display of protein libraries, but similar systems are also being developed using prokaryotic hosts. This review summarizes the field of bacterial surface display with a strong emphasis on library applications for generation of new affinity proteins. The main focus will be on the most recent progress of the work on primarily Escherichia coli, but also on studies using a recently developed system for display on Gram-positive Staphylococcus carnosus. In addition, general strategies for combinatorial protein engineering using cell display are discussed along with the latest developments of new methodologies with comparisons to mainly phage display technology.     

Cytokinesis in bacteria.

Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.