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.

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.

Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents.

SHuffle strains are genetically engineeredEscherichia colistrains that are capable of oxidizing cysteines within proteins to form disulfide bonds. Here we present the complete genome of both the K-12 and B versions of SHuffle strains along with their parental ancestors. These strains have been of significant use to both the general scientific community and the biotech industry, interested in producing novel disulfide-bonded proteins that were hitherto unable to be expressed in standardE. coliexpression strains.

Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase.

AIMS: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. RESULTS: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. INNOVATION AND CONCLUSIONS: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization.

Production of Disulfide-Bonded Proteins in Escherichia coli.

Production of recombinant proteins at high yields in Escherichia coli requires extensive optimization of expression conditions. Production is further complicated for proteins that require specific post-translational modifications for their eventual folding. One common and particularly important post-translational modification is oxidation of the correct pair of cysteines to form a disulfide bond. This unit describes methods to produce disulfide-bonded proteins in E. coli in either the naturally oxidizing periplasm or the cytoplasm of appropriately engineered cells. The focus is on variables key to improving the oxidative folding of disulfide-bonded proteins, with the aim of helping the researcher optimize expression conditions for a protein of interest.

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.

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.

The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.