Promising properties of cytochrome P450 BM3 reconstituted from separate domains by split intein.

Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes.

Split intein converting peptide protein interaction into electrochemically assisted metal ion catalytic signal in the prenatal screening of pediatric epilepsy.

Dravet syndrome is a rare form of epilepsy starting from infancy that can plaque the affected individuals all though his/her life with repeated seizures, and this condition is currently without a complete cure. So prenatal screening of molecular markers of this condition is urgently needed to help couples conceiving new lives to steer clear of this potential danger. And such an assay should ideally be of low cost and could be completed in a point-of-care fashion. This work reports an attempt to construct such an assay using simple peptides in the place of conventional biosensing macro-molecules such as antibodies and enzymes. Specifically, a marker protein of this syndrome can bring the two pieces of a self-splitting peptide "intein" together, which in turn facilitate the formation of metal ion coordination site, recruiting cupric ion to generate catalytically amplified signal readout. Using this method, disease marker protein Nav of this syndrome can be quantitatively detected directly in amniotic fluid samples, and samples associated with potential risk factors such as family history of this syndrome shows statistically evident decrease of this marker protein. These results may promise future application of the proposed method in clinical practice to reduce the social burden of Dravet syndrome by reducing its actual incident rate.

Int&in: A machine learning-based web server for active split site identification in inteins.

Inteins are proteins that excise themselves out of host proteins and ligate the flanking polypeptides in an auto-catalytic process called protein splicing. In nature, inteins are either contiguous or split. In the case of split inteins, the two fragments must first form a complex for the splicing to occur. Contiguous inteins have previously been artificially split in two fragments because split inteins allow for distinct applications than contiguous ones. Even naturally split inteins have been split at unnatural split sites to obtain fragments with reduced affinity for one another, which are useful to create conditional inteins or to study protein-protein interactions. So far, split sites in inteins have been heuristically identified. We developed Int&in, a web server freely available for academic research (https://intein.biologie.uni-freiburg.de) that runs a machine learning model using logistic regression to predict active and inactive split sites in inteins with high accuracy. The model was trained on a dataset of 126 split sites generated using the gp41-1, Npu DnaE and CL inteins and validated using 97 split sites extracted from the literature. Despite the limited data size, the model, which uses various protein structural features, as well as sequence conservation information, achieves an accuracy of 0.79 and 0.78 for the training and testing sets, respectively. We envision Int&in will facilitate the engineering of novel split inteins for applications in synthetic and cell biology.

Novel protein ligase based on dual split intein.

Inteins are unique single-turnover enzymes that can excise themselves from the precursor protein without the aid of any external cofactors or energy. In most cases, inteins are covalently linked with the extein sequences and protein splicing happens spontaneously. In this study, a novel protein ligation system was developed based on two atypical split inteins without cross reaction, in which the large segments of one S1 and one S11 split intein fusion protein acted as a protein ligase, the small segments (only several amino acids long) was fused to the N-extein and C-extein, respectively. The splicing activity was demonstrated in E. coli and in vitro with different extein sequences, which showed ∼15% splicing efficiency in vitro. The protein trans-splicing in vitro was further optimized, and possible reaction explanations were explored. As a proof of concept, we expect this approach to expand the scope of trans-splicing-based protein engineering and provide new clues for intein based protein ligase.

Improved protein splicing through viral passaging.

Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCEIntervening proteins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity.

Split Gp41-1 intein splicing as a model to evaluate the cellular location of the oncosuppressor Maspin in an in vitro model of osteosarcoma.

Inteins are proteins involved in the protein splicing mechanism, an autoprocessing event, where sequences (exteins) separated by inteins become ligated each other after recombination. Two kinds of inteins have been described, contiguous inteins and split inteins. The former ones are transcribed and translated as a single peptide along with their exteins, while the latter are fragmented between two different genes and are transcribed and translated separately. The aim of this study is to establish a method to obtain a fluorescent eukaryotic protein to analyze its cellular localization, using the natural split gp41-1 inteins. We chose natural split inteins due to their distribution in all three domains of life. Two constructs were prepared, one containing the N-terminal split intein along with the N-moiety of the Red Fluorescent Protein (RFP) and a second construct containing the C-terminal of split intein, the C-moiety of RFP and the gene coding for Maspin, a tumor suppressor protein. The trans-splicing was verified by transfecting both N-terminal and C-terminal constructs into mammalian cells. The success of the recombination event was highlighted through the fluorescence produced by reconstituted RFP after recombination, along with the overlap of the red fluorescence produced by recombined RFP and the green fluorescence produced by the hybridization of the recombinant Maspin with a specific antibody. In conclusion, we opted to use this mechanism of recombination to obtain a fluorescent Maspin instead to express a large fusion protein, considering that it could interfere with Maspin's structure and function.

Episomal Vectors for Stable Production of Recombinant Proteins and Engineered Antibodies.

There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein-split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody-siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications.

Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification.

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (ßgal), maltose binding protein (MBP) and beta lactamase (ßlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.

An intein-based biosensor to measure protein stability in vivo.

Biosensors to measure protein stability in vivo are valuable tools for a variety of applications. Previous work has demonstrated that a tripartite design, whereby a protein of interest (POI) is inserted within a reporter, can link POI stability to reporter activity. Inteins are translated within other proteins and excised in a self-mediated protein splicing reaction. Here, we developed a novel folding biosensor where a POI is inserted within an intein, which is subsequently translated within an antibiotic resistance marker. We showed that protein splicing is required for antibiotic resistance and that housing a stable POI within the intein, compared to an unstable variant, results in a 100,000-fold difference in survival. Further, using a fluorescent protein that matures slowly as the POI, we developed a reporter with two simultaneous readouts for protein folding. Finally, we showed that co-expression of GroEL can significantly increase the activity of both reporters, further verifying that protein folding factors can act on the POI in the biosensor. As a whole, our work provides a new twist on the traditional tripartite approach to measuring protein stability in vivo.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone.

In this study, we prepared a functional organic-inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme's terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 âˆ¼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3-11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer.

The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively.

Structural Basis of the Bivalency of the TRPV1 Agonist DkTx.

Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.

Molecular docking and MD simulations reveal protease inhibitors block the catalytic residues in Prp8 intein of Aspergillus fumigatus: a potential target for antimycotics.

Resistance to azoles and amphotericin B especially in Aspergillus fumigatus is a growing concern towards the treatment of invasive fungal infection. At this critical juncture, intein splicing would be a productive, and innovative target to establish therapies against resistant strains. Intein splicing is the central event for the activation of host protein, essential for the growth and survival of various microorganisms including A. fumigatus. The splicing process is a four-step protease-like nucleophilic cascade. Thus, we hypothesise that protease inhibitors would successfully halt intein splicing and potentially restrict the growth of the aforementioned pathogen. Using Rosetta Fold and molecular dynamics simulations, we modelled Prp8 intein structure; resembling classic intein fold with horse shoe shaped splicing domain. To fully comprehend the active site of Afu Prp8 intein, C1, T62, H65, H818, N819 from intein sequences and S820, the first C-extein residue are selected. Molecular docking shows that two FDA-approved drugs, i.e. Lufotrelvir and Remdesivir triphosphate efficiently interact with Prp8 intein from the assortment of 212 protease inhibitors. MD simulation portrayed that Prp8 undergoes conformational change upon ligand binding, and inferred the molecular recognition and stability of the docked complexes. Per-residue decomposition analysis confirms the importance of F: block R802, V803, and Q807 binding pocket in intein splicing domain towards recognition of inhibitors, along with active site residues through strong hydrogen bonds and hydrophobic contacts. However, in vitro and in vivo assays are required to confirm the inhibitory action on Prp8 intein splicing; which may pave the way for the development of new antifungals for A. fumigatus.Communicated by Ramaswamy H. Sarma.

Inteins-mechanism of protein splicing, emerging regulatory roles, and applications in protein engineering.

Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.

Near-Infrared Optogenetic Module for Conditional Protein Splicing.

Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.

Neighboring inteins interfere with one another's homing capacity.

Inteins are mobile genetic elements that invade conserved genes across all domains of life and viruses. In some instances, a single gene will have several intein insertion sites. In Haloarchaea, the minichromosome maintenance (MCM) protein at the core of replicative DNA helicase contains four intein insertion sites within close proximity, where two of these sites (MCM-a and MCM-d) are more likely to be invaded. A haloarchaeon that harbors both MCM-a and MCM-d inteins, Haloferax mediterranei, was studied in vivo to determine intein invasion dynamics and the interactions between neighboring inteins. Additionally, invasion frequencies and the conservation of insertion site sequences in 129 Haloferacales mcm homologs were analyzed to assess intein distribution across the order. We show that the inteins at MCM-a and MCM-d recognize and cleave their respective target sites and, in the event that only one empty intein invasion site is present, readily initiate homing (i.e. single homing). However, when two inteins are present co-homing into an intein-free target sequence is much less effective. The two inteins are more effective when invading alleles that already contain an intein at one of the two sites. Our in vivo and computational studies also support that having a proline in place of a serine as the first C-terminal extein residue of the MCM-d insertion site prevents successful intein splicing, but does not stop recognition of the insertion site by the intein's homing endonuclease.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

A novel protein purification scheme based on salt inducible self-assembling peptides.

BACKGROUND: Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. RESULTS: In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. CONCLUSION: Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.

Backbone Cyclization of Flavin Mononucleotide-Based Fluorescent Protein Increases Fluorescence and Stability.

Flavin mononucleotide-binding proteins or domains emit cyan-green fluorescence under aerobic and anaerobic conditions, but relatively low fluorescence and less thermostability limit their application as reporters. In this work, we incorporated the codon-optimized fluorescent protein from Chlamydomonas reinhardtii with two different linkers independently into the redox-responsive split intein construct, overexpressed the precursors in hyperoxic Escherichia coli SHuffle T7 strain, and cyclized the target proteins in vitro in the presence of the reducing agent. Compared with the purified linear protein, the cyclic protein with the short linker displayed enhanced fluorescence. In contrast, cyclized protein with incorporation of the long linker including the myc-tag and human rhinovirus 3C protease cleavable sequence emitted slightly increased fluorescence compared with the protein linearized with the protease cleavage. The cyclic protein with the short linker also exhibited increased thermal stability and exopeptidase resistance. Moreover, induction of the target proteins in an oxygen-deficient culture rendered fluorescent E. coli BL21 (DE3) cells brighter than those overexpressing the linear construct. Thus, the cyclic reporter can hopefully be used in certain thermophilic anaerobes.

Single-Step Non-Chromatographic Purification of Recombinant Mammalian Proteins Using a Split Intein ELP Tag System.

Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells.