Production and applications of fluorobody from redox-engineered Escherichia coli.

Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E. coli hosts tested, an engineered E. coli B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2-8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets. KEY POINTS: • E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody) • Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal • This platform can be used to produce fluorobodies for numerous purposes.

Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli.

Monoclonal antibodies (mAbs) represent an important platform for the development of biotherapeutic products. Most mAbs are produced in mammalian cells, but several mAbs are made in Escherichia coli, including therapeutic fragments. The NISTmAb is a well-characterized reference material made widely available to facilitate the development of both originator biologics and biosimilars. Here, when expressing NISTmAb from codon-optimized constructs in E. coli (eNISTmAb), a truncated variant of its heavy chain was observed. N-terminal protein sequencing and mutagenesis analyses indicated that the truncation resulted from an internal translation initiation from a GTG codon (encoding Val) within eNISTmAb. Using computational and biochemical approaches, we demonstrate that this translation initiates from a weak Shine-Dalgarno sequence and is facilitated by a putative ribosomal protein S1-binding site. We also observed similar internal initiation in the mAb adalimumab (the amino acid sequence of the drug Humira) when expressed in E. coli Of note, these internal initiation regions were likely an unintended result of the codon optimization for E. coli expression, and the amino acid pattern from which it is derived was identified as a Pro-Ser-X-X-X-Val motif. We discuss the implications of our findings for E. coli protein expression and codon optimization and outline possible strategies for reducing the likelihood of internal translation initiation and truncated product formation.

Improved production of Humira antibody in the genetically engineered Escherichia coli SHuffle, by co-expression of human PDI-GPx7 fusions.

Microbial production of antibodies offers the promise of cheap, fast, and efficient production of antibodies at an industrial scale. Limiting this capacity in prokaryotes is the absence of the post-translational machinery, present in dedicated antibody producing eukaryotic cell lines, such as B cells. There has been few and limited success in producing full-length, correctly folded, and assembled IgG in the cytoplasm of prokaryotic cell lines. One such success was achieved by utilizing the genetically engineered Escherichia coli strain SHuffle with an oxidative cytoplasm. Due to the genetic disruption of reductive pathways, SHuffle cells are under constant oxidative stress, including increased levels of hydrogen peroxide (H2O2). The oxidizing capacity of H2O2 was linked to improved disulfide bond formation, by expressing a fusion of two endoplasmic reticulum-resident proteins, the thiol peroxidase GPx7 and the protein disulfide isomerase, PDI. In concert, these proteins mediate disulfide transfer from H2O2 to target proteins via PDI-Gpx7 fusions. The potential of this new strain was tested with Humira, a blockbuster antibody usually produced in eukaryotic cells. Expression results demonstrate that the new engineered SHuffle strain (SHuffle2) could produce Humira IgG four-fold better than the parental strain, both in shake-flask and in high-density fermentation. These preliminary studies guide the field in genetically engineering eukaryotic redox pathways in prokaryotes for the production of complex macromolecules. KEY POINTS: • A eukaryotic redox pathway was engineered into the E. coli strain SHuffle in order to improve the yield of the blockbuster antibody Humira. • The best peroxidase-PDI fusion was selected using bioinformatics and in vivo studies. • Improved yields of Humira were demonstrated at shake-flask and high-density fermenters.

A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo.

Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were coexpressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide-bond formation in a range of substrate proteins, in a manner dependent on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way to unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid-bilayer context.

Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli.

A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25 nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.

Visualizing Bacterial Colony Morphologies Using Time-Lapse Imaging Chamber MOCHA.

Capturing microbial growth on a macroscopic scale is of great importance to further our understanding of microbial life. However, methods for imaging microbial life on a scale of millimeters to centimeters are often limited by designs that have poor environmental control, resulting in dehydration of the agar plate within just a few days. Here, we created MOCHA (microbial chamber), a simple but effective chamber that allows users to study microbial growth for extended periods (weeks) in a stable environment. Agar hydration is maintained with a double-decker design, in which two glass petri dishes are connected by a wick, allowing the lower plate to keep the upper plate hydrated. This flexible chamber allows the observation of a variety of microbiological phenomena, such as the growth and development of single bacterial and fungal colonies, interspecies interactions, swarming motility, and pellicle formation.IMPORTANCE Detailed study of microbial life on the colony scale of millimeters to centimeters has been lagging considerably behind microscopic inspection of microbes. One major reason for this is the lack of inexpensive instrumentation that can reproducibly capture images in a controlled environment. In this study, we present the design and use of a unique chamber that was used to produce several time-lapse movies that aimed to capture the diversity of microbial colony phenotypes over long periods.

Aeropyrum pernix membrane topology of protein VKOR promotes protein disulfide bond formation in two subcellular compartments.

Disulfide bonds confer stability and activity to proteins. Bioinformatic approaches allow predictions of which organisms make protein disulfide bonds and in which subcellular compartments disulfide bond formation takes place. Such an analysis, along with biochemical and protein structural data, suggests that many of the extremophile Crenarachaea make protein disulfide bonds in both the cytoplasm and the cell envelope. We have sought to determine the oxidative folding pathways in the sequenced genomes of the Crenarchaea, by seeking homologues of the enzymes known to be involved in disulfide bond formation in bacteria. Some Crenarchaea have two homologues of the cytoplasmic membrane protein VKOR, a protein required in many bacteria for the oxidation of bacterial DsbAs. We show that the two VKORs of Aeropyrum pernix assume opposite orientations in the cytoplasmic membrane, when expressed in E. coli. One has its active cysteines oriented toward the E. coli periplasm (ApVKORo) and the other toward the cytoplasm (ApVKORi). Furthermore, the ApVKORo promotes disulfide bond formation in the E. coli cell envelope, while the ApVKORi promotes disulfide bond formation in the E. coli cytoplasm via a co-expressed archaeal protein ApPDO. Amongst the VKORs from different archaeal species, the pairs of VKORs in each species are much more closely related to each other than to the VKORs of the other species. The results suggest two independent occurrences of the evolution of the two topologically inverted VKORs in archaea. Our results suggest a mechanistic basis for the formation of disulfide bonds in the cytoplasm of Crenarchaea.

Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag.

UNLABELLED: The use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryote Escherichia coli. We show that functional protein fusions of the HaloTag can be detected both in vivo and in vitro when expressed within the cytoplasmic or periplasmic compartments of E. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications. IMPORTANCE: Visualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryote Escherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

SAS: Split antibiotic selection for identifying chaperones that improve protein solubility.

Background: Heterologous expression of active, native-folded protein in Escherichia coli is critical in both academic research and biotechnology settings. When expressing non-native recombinant proteins in E. coli, obtaining soluble and active protein can be challenging. Numerous techniques can be used to enhance a proteins solubility, and largely focus on either altering the expression strain, plasmid vector features, growth conditions, or the protein coding sequence itself. However, there is no one-size-fits-all approach for addressing issues with protein solubility, and it can be both time and labor intensive to find a solution. An alternative approach is to use the co-expression of chaperones to assist with increasing protein solubility. By designing a genetic system where protein solubility is linked to viability, the appropriate protein folding factor can be selected for any given protein of interest. To this end, we developed a Split Antibiotic Selection (SAS) whereby an insoluble protein is inserted in-frame within the coding sequence of the hygromycin B resistance protein, aminoglycoside 7″-phosphotransferase-Ia (APH(7″)), to generate a tripartite fusion. By creating this tripartite fusion with APH(7″), the solubility of the inserted protein can be assessed by measuring the level of hygromycin B resistance of the cells. Results: We demonstrate the functionality of this system using a known protein and co-chaperone pair, the human mitochondrial Hsp70 ATPase domain (ATPase70) and its co-chaperone human escort protein (Hep). Insertion of the insoluble ATPase70 within APH(7'') renders the tripartite fusion insoluble and results in sensitivity to hygromycin B. Antibiotic resistance can be rescued by expression of the co-chaperone Hep which assists in the folding of the APH(7'')-ATPase70-APH(7'') tripartite fusion and find that cellular hygromycin B resistance correlates with the total soluble fusion protein. Finally, using a diverse chaperone library, we find that SAS can be used in a pooled genetic selection to identify chaperones capable of improving client protein solubility. Conclusions: The tripartite APH(7'') fusion links the in vivo solubility of the inserted protein of interest to hygromycin B resistance. This construct can be used in conjunction with a chaperone library to select for chaperones that increase the solubility of the inserted protein. This selection system can be applied to a variety of client proteins and eliminates the need to individually test chaperone-protein pairs to identify those that increase solubility.

Bridging the gap with bacterial art.

Living art made with bacteria is gaining global attention, spreading from laboratories into the public domain: from school STEAM (Science, Technology, Engineering, the Arts, and Mathematics) events to art galleries, museums, community labs, and ultimately to the studios of microbial artists. Bacterial art is a synthesis of science and art that can lead to developments in both fields. Through the 'universal language of art', many social and preconceived ideas-including abstract scientific concepts-can be challenged and brought to the public attention in a unique way. By using bacteria to create publicly accessible art pieces, the barriers between humans and microbes can be lessened, and the artificial separation of the fields of science and art may be brought one step closer. Here, we document the history, impact, and current moment in the field of microbiologically inspired art for the benefit of educators, students, and the interested public. We provide a comprehensive historical background and examples of ancient bacterial art from cave paintings to uses in modern synthetic biology, a simple protocol for conducting bacterial art in a safe and responsible manner, a discussion of the artificial separation of science and art, and the future implications of art made from living microbes.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Production of disulfide-bonded proteins in Escherichia coli.

Disulfide bonds are covalent bonds formed post-translationally by the oxidation of a pair of cysteines. A disulfide bond can serve structural, catalytic, and signaling roles. However, there is an inherent problem to the process of disulfide bond formation: mis-pairing of cysteines can cause misfolding, aggregation and ultimately result in low yields during protein production. Recent developments in the understanding of the mechanisms involved in the formation of disulfide bonds have allowed the research community to engineer and develop methods to produce multi-disulfide-bonded proteins to high yields. This review attempts to highlight the mechanisms responsible for disulfide bond formation in Escherichia coli, both in its native periplasmic compartment in wild-type strains and in the genetically modified cytoplasm of engineered strains. The purpose of this review is to familiarize the researcher with the biological principles involved in the formation of disulfide-bonded proteins with the hope of guiding the scientist in choosing the optimum expression system.

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm.

BACKGROUND: Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. RESULTS: We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. CONCLUSIONS: This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using SHuffle strains.

TrbB from conjugative plasmid F is a structurally distinct disulfide isomerase that requires DsbD for redox state maintenance.

TrbB, a periplasmic protein encoded by the conjugative plasmid F, has a predicted thioredoxin-like fold and possesses a C-X-X-C redox active site motif. TrbB may function in the conjugative process by serving as a disulfide bond isomerase, facilitating proper folding of a subset of F-plasmid-encoded proteins in the periplasm. Previous studies have demonstrated that a ΔtrbB F plasmid in Escherichia coli lacking DsbC(E.coli), its native disulfide bond isomerase, experiences a 10-fold decrease in mating efficiency but have not provided direct evidence for disulfide bond isomerase activity. Here we demonstrate that trbB can partially restore transfer of a variant of the distantly related R27 plasmid when both chromosomal and plasmid genes encoding disulfide bond isomerases have been disrupted. In addition, we show that TrbB displays both disulfide bond isomerase and reductase activities on substrates not involved in the conjugative process. Unlike canonical members of the disulfide bond isomerase family, secondary structure predictions suggest that TrbB lacks both an N-terminal dimerization domain and an α-helical domain found in other disulfide bond isomerases. Phylogenetic analyses support the conclusion that TrbB belongs to a unique family of plasmid-based disulfide isomerases. Interestingly, although TrbB diverges structurally from other disulfide bond isomerases, we show that like those isomerases, TrbB relies on DsbD from E. coli for maintenance of its C-X-X-C redox active site motif.

In vivo oxidative protein folding can be facilitated by oxidation-reduction cycling.

Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis, a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region; that it is a very active reductase but a poor isomerase; and that it fully complements dsbC. These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.

Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation.

Protein disulfide bond formation contributes to the folding and activity of many exported proteins in bacteria. However, information about disulfide bond formation is limited to only a few bacterial species. We used a multifaceted bioinformatic approach to assess the capacity for disulfide bond formation across this biologically diverse group of organisms. We combined data from a cysteine counting method, in which a significant bias for even numbers of cysteine in proteins is taken as an indicator of disulfide bond formation, with data on the presence of homologs of known disulfide bond formation enzymes. These combined data enabled us to make predictions about disulfide bond formation in the cell envelope across bacterial species. Our bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a disulfide bond formation pathway present in several major bacterial phyla.