Strategies and opportunities for engineering antifungal peptides for therapeutic applications.

Antifungal peptides (AFPs) are widely described as promising prospects to treat and prevent fungal infections, though they are far less studied than their antibacterial counterparts. Although promising, AFPs have practical limitations that have hindered their use as therapeutics. Rational design and combinatorial engineering are powerful protein engineering strategies with much potential to address the limitations of AFPs by designing peptides with improved physiochemical and biological characteristics. We examine how rational design and combinatorial engineering approaches have already been used to improve the properties of AFPs and propose key opportunities for applying these strategies to push the design and application of AFPs forward.

Quantifying the Antifungal Activity of Peptides Against Candida albicans.

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Capsules with bacteria and fungi in distinct compartments: A platform for studying microbes from different kingdoms and their cross-communication.

Recently, we have created 'artificial cells' with an architecture mimicking that of typical eukaryotic cells. Our design uses common biopolymers like alginate and chitosan to create multi-compartment capsules (MCCs) via oil-free microfluidics. MCCs (~ 500 µm in diameter) can be engineered with multiple inner compartments, each with a distinct payload. This mimics the distinct organelles in eukaryotic cells, each of which has unique properties. In this study, we encapsulate microbial cells from two distinct kingdoms - Pseudomonas aeruginosa (bacteria) and Candida albicans (fungi) - in the inner compartments of MCCs. The two microbes are commonly found in biofilms at sites of infection in humans. We first demonstrate that the MCC can serve as a simple platform to observe the comparative growth of the cells in real time. Unlike typical co-culture in solution or on agar plates, the cells can grow in their own compartments without direct physical contact. Moreover, the hydrogel matrix in the compartments mimics the three-dimensional (3-D) environment that cells naturally encounter during their growth. Small molecules added to the solution are shown to permeate through the capsule walls and affect cell growth: for example, cationic surfactants inhibit the fungi but not the bacteria. Conversely, low pH and kanamycin inhibit the bacteria but not the fungi. Also, when the bacteria are present in adjacent compartments, the fungal cells mostly stay in a yeast morphology, meaning as spheroidal cells. In contrast, in the absence of the bacteria, the fungi transition into hyphae, i.e., long multicellular filaments. The inhibition of this morphological switch in fungal cells is shown to be induced by signaling molecules (specifically, the quorum sensing autoinducer-1 or AI-1) secreted by the bacteria. Thus, the MCC platform can also be used to detect cross-kingdom signaling between the compartmentalized microbes.

Histatin 5 variant reduces Candida albicans biofilm viability and inhibits biofilm formation.

Candida albicans is a commensal organism and opportunistic pathogen that can form biofilms that colonize surfaces of medical devices, such as implants, catheters, and dentures. Compared to planktonic C. albicans cells, cells in biofilms exhibit increased resistance to treatment. Histatin 5 (Hst-5) is an antimicrobial peptide that is natively secreted by human salivary glands and has strong antifungal activity against C. albicans. However, C. albicans produces secreted aspartic proteases (Saps) that can cleave and inactivate Hst-5, limiting its antifungal properties. We previously showed that residue substitutions K11R and K17R within Hst-5 improve its antifungal activity and prevent proteolytic degradation by Saps when treating planktonic C. albicans. Here, we investigated the use of the K11R-K17R peptide as an alternative therapeutic against C. albicans biofilms by assessing its ability to reduce viability of pre-formed biofilms and to inhibit the formation of biofilms and showed that K11R-K17R had improved activity compared to Hst-5. Based on these results, we incorporated K11R-K17R and Hst-5 into polyelectrolyte multilayer (PEM) surface coatings and demonstrated that films functionalized with K11R-K17R reduced the formation of C. albicans biofilms. Our results demonstrate the therapeutic potential of the K11R-K17R Hst-5 variant in preventing and treating biofilms.

Online capillary electrophoresis - mass spectrometry analysis of histatin-5 and its degradation products.

Histatin-5 (Hst-5) is a human salivary peptide with antibacterial and antifungal activities. Thorough characterization and reliable quantification of Hst-5 and its degradation products are essential for understanding the Hst-5 degradation pathway. Due to the highly basic and strong cationic nature of the Hst-5 peptide, the quantitative analysis of Hst-5 and its degradation forms by online mass spectrometry remains challenging. Here, we adopt a recently developed electrokinetically pumped sheath liquid capillary electrophoresis - mass spectrometry (CE-MS) coupling technology, and successfully apply it for the analysis of Hst-5 and its degradation products. Our CE-MS method is demonstrated to be robust and quantitative. This novel analytical platform is reproducible and free of sample carryover. The efficacy of this method is demonstrated with a kinetic study of Hst-5 degradation by Sap9, a secreted aspartic peptidase. Our work demonstrates the potential of online CE-MS as a powerful approach for characterizing highly basic peptides.

Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis.

Histatin 5 (Hst-5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst-5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single-residue substitutions can significantly affect the proteolytic resistance of Hst-5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single-residue modifications can be combined in a single peptide, K11R-K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single-residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap-dependent effects on degradation. Both single- and double-substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.

Evaluation of the Antifungal and Wound-Healing Properties of a Novel Peptide-Based Bioadhesive Hydrogel Formulation.

Oral candidiasis (OC) caused by the fungal pathogen Candida albicans is the most common opportunistic infection in immunocompromised populations. The dramatic increase in resistance to common antifungal agents has emphasized the importance of identifying alternative therapeutic options. Antimicrobial peptides have emerged as promising drug candidates due to their antimicrobial properties; specifically, histatin-5 (Hst-5), a peptide naturally produced and secreted by human salivary glands, has demonstrated potent activity against C. albicans However, as we previously demonstrated vulnerability for Hst-5 to proteolysis by C. albicans proteolytic enzymes at specific amino acid residues, a new variant (K11R-K17R) was designed with amino acid substitutions at the identified cleavage sites. The new resistant peptide demonstrated no cytotoxicity to erythrocytes or human oral keratinocytes. To evaluate the potential of the new peptide for clinical application, we utilized our FDA-approved polymer-based bioadhesive hydrogel as a delivery system and developed a therapeutic formulation specifically designed for oral topical application. The new formulation was demonstrated to be effective against C. albicans strains resistant to the traditional antifungals, and the in vitro therapeutic efficacy was found to be comparable to that of the common topical antifungal agents in clinical use. Importantly, in addition to its antifungal properties, our findings also demonstrated that the new peptide variant induces cell proliferation and rapid cell migration of human oral keratinocytes, indicative of wound healing properties. The findings from this study support the progression of the novel formulation as a therapeutic agent against oral candidiasis, as well as a therapeutic modality for promoting wound healing.

Expression of Cell-Penetrating Peptides Fused to Protein Cargo.

Cell-penetrating peptides (CPPs) are short peptides that can cross cell membranes. CPPs enable the delivery of biomolecules into cells and can act as drug-delivery vectors. Because recombinant production of CPPs as fusions to protein "cargo" leads to low yields for some CPP-cargo fusions, approaches to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions in Escherichia coli for fusions of CPPs (SynB, histatin-5, and MPG) to the cargo proteins biotin carboxyl carrier protein, maltose-binding protein, and green fluorescent protein. We used Western blotting to evaluate induction temperatures of 37, 30, and 20°C, and induction times of 6, 10, and 24 h. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37°C for 6 and 10 h led to the highest levels of expression for the different CPP-cargo constructs. The improvements in expression of CPP-cargo fusions will allow higher yields of CPP-cargo fusions for studies of their translocation into cells.

Effect of linkers on immobilization of scFvs with biotin-streptavidin interaction.

Single-chain variable fragment antibodies (scFvs) are attractive for use in applications that require high specificity and binding to a target, such as biosensors. Previously, we demonstrated that a variety of scFvs can be immobilized onto a streptavidin surface through in vivo biotinylation of the biotin carboxyl carrier protein (BCCP) or smaller AviTag fused to the scFvs. However, the BCCP constructs showed better immobilization than the AviTag constructs. In this work, we investigated whether the discrepancy between the biotinylation tags could be alleviated by incorporating a flexible (G4 S)n linker of varying lengths or a rigid (EA3 K)3 linker between the biotinylation tags and the scFvs scFv13R4 and scFv5. Fusion of the (G4 S)5 linker or the (G4 S)3 linker to the AviTag construct of scFv13R4 or scFv5, respectively, and fusion of the (EA3 K)3 linkers to the AviTag constructs of both scFvs enhanced immobilization. Meanwhile, the robust immobilization of the BCCP construct of the scFv constructs remained unaffected. The positive to neutral effects of the linkers, with no adverse effects, make them beneficial tools to incorporate into fusion proteins that show poor immobilization without a linker.

Protein Labeling in Live Cells for Immunological Applications.

Protein labeling is often an important aspect of immunological experiments, as it allows observation of cellular processes, including protein synthesis and trafficking. Many protein labeling methods require permeabilization and fixation of cells, damaging the cells and preventing observation of processes in real time. However, a number of bioconjugation techniques allow protein labeling inside living cells to allow visualization of cellular processes as they occur and to facilitate retrieval of desired proteins. In this Topical Review, we describe bioconjugation methods that allow specific labeling of intracellular proteins of interest and discuss their applications to immunological studies. We focus on protein fusions, biotinylation, fluorescein arsenical helix binder (FlAsH) and resorufin arsenical helix binder (ReAsH) labeling, and tetrazine ligation.

Secondary structure of cell-penetrating peptides during interaction with fungal cells.

Cell-penetrating peptides (CPPs) are peptides that cross cell membranes, either alone or while carrying molecular cargo. Although their interactions with mammalian cells have been widely studied, much less is known about their interactions with fungal cells, particularly at the biophysical level. We analyzed the interactions of seven CPPs (penetratin, Pep-1, MPG, pVEC, TP-10, MAP, and cecropin B) with the fungal pathogen Candida albicans using experiments and molecular simulations. Circular dichroism (CD) of the peptides revealed a structural transition from a random coil or weak helix to an α-helix occurs for all peptides when the solvent is changed from aqueous to hydrophobic. However, CD performed in the presence of C. albicans cells showed that proximity to the cell membrane is not necessarily sufficient to induce this structural transition, as penetratin, Pep-1, and MPG did not display a structural shift in the presence of cells. Monte Carlo simulations were performed to further probe the molecular-level interaction with the cell membrane, and these simulations suggested that pVEC, TP-10, MAP, and cecropin B strongly penetrate into the hydrophobic domain of the membrane lipid bilayer, inducing a transition to an α-helical conformation. In contrast, penetratin, Pep-1 and MPG remained in the hydrophilic region without a shift in conformation. The experimental data and MC simulations combine to explain how peptide structure affects their interaction with cells and their mechanism of translocation into cells (direct translocation vs. endocytosis). Our work also highlights the utility of combining biophysical experiments, biological experiments, and molecular modeling to understand biological phenomena.

Engineering improved variants of the antifungal peptide histatin 5 with reduced susceptibility to Candida albicans secreted aspartic proteases and enhanced antimicrobial potency.

Candida albicans is an opportunistic fungal pathogen and a commensal organism that commonly colonizes mucosal surfaces, including those inside the human mouth. To help control C. albicans, human saliva contains the antifungal peptide histatin 5 (Hst-5), which has strong antifungal activity against C. albicans. However, the pathogen produces secreted aspartic proteases (Saps) that cleave Hst-5 at lysine residues and eliminate its antifungal properties. We designed variants of Hst-5 with its lysine residues substituted with arginine or leucine to evaluate the effect on proteolysis by Saps. We found site-, residue-, and Sap-dependent effects from single amino acid substitutions. The K17R and K17L modifications led to dramatic results, with over 77% and 100% intact peptide remaining after incubation with Sap9 and Sap2, respectively, compared to 47% and 61% of Hst-5. This decrease in proteolysis was accompanied by a reduction in cleavage on the C-terminal side of K17, suggesting the Saps prefer lysine at K17 for cleavage. Incubation with C. albicans cells and culture supernatant corroborated the results with purified Saps and highlighted their biological relevance. The modifications to Hst-5 do not diminish the antifungal activity of Hst-5, and, in fact, the K17R, K17L, and K11R peptides retained significantly more antifungal activity after treatment with Saps than Hst-5. Our results indicate that single amino acid modifications drastically impact both proteolysis at the modification sites and the overall level of proteolysis of the peptide, demonstrating the potential of designing peptides for resistance to proteolysis as a means for improving therapeutic efficacy.

Translocation of cell-penetrating peptides into Candida fungal pathogens.

Cell-penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)3 K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration-dependent manner, and an additional peptide (TP-10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)3 K, and TP-10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.

Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury.

Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin ß1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for ß-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin ß1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin ß-mediated nuclear import. We also observed increased importin ß1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS.

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

Effect of a Flexible Linker on Recombinant Expression of Cell-Penetrating Peptide Fusion Proteins and Their Translocation into Fungal Cells.

Cell-penetrating peptides (CPPs) are a class of small peptides that are able to cross cell membranes via direct translocation or endocytosis. They have been widely used to deliver tethered bioactive molecules to cells, but recombinantly producing CPPs as fusions to protein cargo leads to low yields. We used Escherichia coli cells to recombinantly produce genetic fusions of NPFSD (derived from a yeast endocytosis signal) and pVEC (derived from a murine vascular endothelium cadherin) to the N-terminus of green fluorescent protein (GFP) with and without a flexible glycine-serine linker between the CPP and GFP. The flexible linker improved the expression of the NPFSD construct and the pVEC construct, resulting in a 24.5 % improvement in yield for the NPFSD fusion and a 50.0 % improvement in yield for the pVEC fusion. The linker did not diminish the ability of the fusions to translocate into the fungal pathogen Candida albicans, and the translocation of the NPFSD constructs actually increased by 58 % at 10 min. Moreover, the toxicity of the fusions towards C. albicans was not affected by the incorporation of the linker. These results illustrate the utility of including a linker for CPP-cargo fusions and the potential of NPFSD and pVEC fusions for use in delivering protein cargo to C. albicans.

A simple and robust approach to immobilization of antibody fragments.

Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli. The biotinylated scFvs were immobilized onto streptavidin-coated plates directly from cell lysates, and immobilization was detected through enzyme-linked immunosorbent assays. All scFvs fusions were successfully immobilized, and scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The ability of immobilized scFvs to bind antigens was confirmed using scFv13R4 and scFvD10 with their respective targets ß-galactosidase and bacteriophage lambda head protein D (gpD). The immobilized scFv13R4 bound to ß-galactosidase at the same level for both biotinylation tags when the surface was saturated with the scFv, and immobilized scFvs retained their functionality for at least 100days after immobilization. The simplicity and robustness of our method make it a promising approach for future applications that require antibody fragment immobilization.

Development and In Vivo Evaluation of a Novel Histatin-5 Bioadhesive Hydrogel Formulation against Oral Candidiasis.

Oral candidiasis (OC), caused by the fungal pathogen Candida albicans, is the most common opportunistic infection in HIV(+) individuals and other immunocompromised populations. The dramatic increase in resistance to common antifungals has emphasized the importance of identifying unconventional therapeutic options. Antimicrobial peptides have emerged as promising candidates for therapeutic intervention due to their broad antimicrobial properties and lack of toxicity. Histatin-5 (Hst-5) specifically has exhibited potent anticandidal activity indicating its potential as an antifungal agent. To that end, the goal of this study was to design a biocompatible hydrogel delivery system for Hst-5 application. The bioadhesive hydroxypropyl methylcellulose (HPMC) hydrogel formulation was developed for topical oral application against OC. The new formulation was evaluated in vitro for gel viscosity, Hst-5 release rate from the gel, and killing potency and, more importantly, was tested in vivo in our mouse model of OC. The findings demonstrated a controlled sustained release of Hst-5 from the polymer and rapid killing ability. Based on viable C. albicans counts recovered from tongues of treated and untreated mice, three daily applications of the formulation beginning 1 day postinfection with C. albicans were effective in protection against development of OC. Interestingly, in some cases, Hst-5 was able to clear existing lesions as well as associated tissue inflammation. These findings were confirmed by histopathology analysis of tongue tissue. Coupled with the lack of toxicity as well as anti-inflammatory and wound-healing properties of Hst-5, the findings from this study support the progression and commercial feasibility of using this compound as a novel therapeutic agent.

Structure-activity relationships among antifungal nylon-3 polymers: identification of materials active against drug-resistant strains of Candida albicans.

Fungal infections are a major challenge to human health that is heightened by pathogen resistance to current therapeutic agents. Previously, we were inspired by host-defense peptides to develop nylon-3 polymers (poly-ß-peptides) that are toxic toward the fungal pathogen Candida albicans but exert little effect on mammalian cells. Based on subsequent analysis of structure-activity relationships among antifungal nylon-3 polymers, we have now identified readily prepared cationic homopolymers active against strains of C. albicans that are resistant to the antifungal drugs fluconazole and amphotericin B. These nylon-3 polymers are nonhemolytic. In addition, we have identified cationic-hydrophobic copolymers that are highly active against a second fungal pathogen, Cryptococcus neoformans, and moderately active against a third pathogen, Aspergillus fumigatus.

Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.

A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Through the use of just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.