Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.     

Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.     

Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain

Green fluorescent protein (GFP) has been used to report protein folding by correlating solubility with fluorescence. In a GFP fusion protein, an upstream aggregation-prone domain can disrupt de novo folding of the GFP domain in Escherichia coli, resulting in a loss of fluorescence. Previously, we showed that prevention of misfolding of the upstream aggregation-prone domain by a coupled folding and binding interaction during protein synthesis restored both GFP fluorescence and solubility. Since molecular chaperones often fold nascent polypeptides through a bind-and-release interaction, the question remains whether the chaperone interaction with the upstream aggregation-prone domain enhances GFP fluorescence. Here, we demonstrate that a significant increase in GFP fluorescence occurred only when appropriate chaperones that recognized the aggregation-prone protein and helped its folding were co-expressed. A possible correlation between GFP fluorescence and the productive folding by chaperones is proposed. This study may provide a general strategy for identifying chaperones specific for difficult-to-fold proteins.     

A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis

We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.     

Structural and thermodynamic analysis of the GFP:GFP-nanobody complex

The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.     

Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein

Cystine-knot miniproteins (knottins) are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI) as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering.     

Protein Design through Systematic Catalytic Loop Exchange in the (β/α)8 Fold

Protein engineering by directed evolution has proven effective in achieving various functional modifications, but the well-established protocols for the introduction of variability, typically limited to random point mutations, seriously restrict the scope of the approach. In an attempt to overcome this limitation, we sought to explore variant libraries with richer diversity at regions recognized as functionally important through an exchange of natural components, thus combining design with combinatorial diversity. With this approach, we expected to maintain interactions important for protein stability while directing the introduction of variability to areas important for catalysis.Our strategy consisted in loop exchange over a (β/α)₈ fold. Phosphoribosylanthranilate isomerase was chosen as scaffold, and we investigated its tolerance to loop exchange by fusing variant libraries to the chloramphenicol acetyl transferase coding gene as an in vivo folding reporter. We replaced loops 2, 4, and 6 of phosphoribosylanthranilate isomerase with loops of varied types and sizes from enzymes sharing the same fold.To allow for a better structural fit, saturation mutagenesis was adopted at two amino acid positions preceding the exchanged loop. Our results showed that 30% to 90% of the generated mutants in the different libraries were folded. Some variants were selected for further characterization after removal of chloramphenicol acetyl transferase gene, and their stability was studied by circular dichroism and fluorescence spectroscopy. The sequences of 545 clones show that the introduction of variability at “hinges” connecting the loops with the scaffold exhibited a noticeable effect on the appearance of folded proteins. Also, we observed that each position accepted foreign loops of different sizes and sequences.We believe our work provides the basis of a general method of exchanging variably sized loops within the (β/α)₈ fold, affording a novel starting point for the screening of novel activities as well as modest diversions from an original activity.     

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.     

Flowering buds of globular proteins: transpiring simplicity of protein organization

Structural and functional complexity of proteins is dramatically reduced to a simple linear picture when the laws of polymer physics are considered. A basic unit of the protein structure is a nearly standard closed loop of 25-35 amino acid residues, and every globular protein is built of consecutively connected closed loops. The physical necessity of the closed loops had been apparently imposed on the early stages of protein evolution. Indeed, the most frequent prototype sequence motifs in prokaryotic proteins have the same sequence size, and their high match representatives are found as closed loops in crystallized proteins. Thus, the linear organization of the closed loop elements is a quintessence of protein evolution, structure and folding.     

Green fluorescent protein as a scaffold for intracellular presentation of peptides.

Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence.     

Rapid protein-folding assay using green fluorescent protein.

Formation of the chromophore of green fluorescent protein (GFP) depends on the correct folding of the protein. We constructed a "folding reporter" vector, in which a test protein is expressed as an N-terminal fusion with GFP. Using a test panel of 20 proteins, we demonstrated that the fluorescence of Escherichia coli cells expressing such GFP fusions is related to the productive folding of the upstream protein domains expressed alone. We used this fluorescent indicator of protein folding to evolve proteins that are normally prone to aggregation during expression in E. coli into closely related proteins that fold robustly and are fully soluble and functional. This approach to improving protein folding does not require functional assays for the protein of interest and provides a simple route to improving protein folding and expression by directed evolution.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein–protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold

The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. We diversified positions on a β-sheet and surface loops that together form a concave surface. The new library produced binding proteins (termed "monobodies") to multiple target proteins, generally with similar efficacy as the original, loop-focused library. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics.     

Understanding mechanisms governing protein-protein interactions from synthetic binding interfaces

Recent advances in methodologies and design of combinatorial library selection have enabled comprehensive characterization of sequence space for protein-protein interaction interfaces and generation of fully synthetic binding interfaces. By exhaustively introducing and quantitatively analyzing mutations in natural interfaces, new insights into their molecular architecture and plasticity have emerged. Minimalist combinatorial libraries based on a restricted amino acid code have produced synthetic interfaces that rival natural ones using a different set of rules. A two amino acid code composed of just tyrosine and serine in the context of antibody CDR loops is sufficient to produce high affinity and specific interactions with different classes of protein targets. Structural analyses highlight the dominant role of Tyr in forming productive interactions and demonstrate the dominance of conformational diversity over chemical diversity in producing na?ve binding interfaces. Synthetic binding proteins are beginning to be used as a powerful crystallization tool to attack important structural biology problems that are recalcitrant to crystallization using traditional methods.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein-protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

FRET-based in vivo screening for protein folding and increased protein stability

Fluorescence resonance energy transfer (FRET) was used to establish a novel in vivo screening system that allows rapid detection of protein folding and protein variants with increased thermodynamic stability in the cytoplasm of Escherichia coli. The system is based on the simultaneous fusion of the green fluorescent protein (GFP) to the C terminus of a protein X of interest, and of blue-fluorescent protein (BFP) to the N terminus of protein X. Efficient FRET from BFP to GFP in the ternary fusion protein is observed in vivo only when protein X is folded and brings BFP and GFP into close proximity, while FRET is lost when BFP and GFP are far apart due to unfolding or intracellular degradation of protein X. The screening system was validated by identification of antibody V(L) intradomains with increased thermodynamic stabilities from expression libraries after random mutagenesis, bacterial cell sorting, and colony screening.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.     

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 beta-lactamase and its inhibitory protein as our model protein-ligand system. 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cell biology.     

Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2

The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were suppressed efficiently by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of six, eight and ten amino acid insertions. MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, and they encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity-selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.