High cytoplasmic expression in E. coli, purification, and in vitro refolding of a single chain Fv antibody fragment against the hepatitis B surface antigen.

A single-chain Fv (scFv) antibody fragment against the hepatitis B surface antigen (HBsAg) was expressed in Escherichia coli in the form of two independent fusion proteins, with either 60 ('long') or 27 ('short') amino acid N-terminal encoding sequences related to human interleukin-2. Both fusion proteins were expressed insolubly and at high levels in the bacterial cytoplasm (approximately 30% of total bacterial protein in MM294 cells at a laboratory scale). When recombinant cells were cultured in 5-1 fermentors, expression and optical density increased 2- and 4-fold, respectively, compared to a previous periplasmic insoluble version of the same anti HBsAg scFv. After extraction and solubilization in urea, the cytoplasmic scFvs were purified using immobilized metal ion affinity chromatography, followed by DTT treatment, and refolding by dialysis against a basic pH buffer containing EDTA. The refolded scFvs recognized the recombinant HBsAg in ELISA. Results of an ELISA where antigen affinity chromatography repurified scFvs were used as standards, indicated that refolding efficiencies were high: 56.2% for the 'short' fusion scFv, and 50.6% for the 'long' fusion scFv. Corrected final yields of active scFv were 30.3 and 27.3 mg l-1, respectively, for the aforementioned fusion proteins, 5-6 times better than those reported for the periplasmic scFv variant.     

Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin

In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.     

From transcriptional landscapes to the identification of biomarkers for robustness

Background

Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis.

Results

The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody.

Conclusions

Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of pseudotype VLPs was successful and allowed generation of multivalent scFv-Fc proteins with high VLY-neutralizing potency. Our study demonstrated for the first time that large recombinant antibody molecule fused with hamster polyomavirus VP2 protein and co-expressed with VP1 protein in the form of pseudotype VLPs was properly folded and exhibited strong antigen-binding activity. The current study broadens the potential of recombinant VLPs as a highly efficient carrier for functionally active complex proteins.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus.

HNK20 is a mouse monoclonal IgA that binds to the F glycoprotein of respiratory syncytial virus (RSV) and neutralizes the virus, both in vitro and in vivo. The single-chain antibody fragment (scFv) derived from HNK20 is equally active and has allowed us to assess rapidly the effect of mutations on affinity and antiviral activity. Humanization by variable domain resurfacing requires that surface residues not normally found in a human Fv be mutated to the expected human amino acid, thereby eliminating potentially immunogenic sites. We describe the construction and characterization of two humanized scFvs, hu7 and hu10, bearing 7 and 10 mutations, respectively. Both molecules show unaltered binding affinities to the RSV antigen (purified F protein) as determined by ELISA and surface plasmon resonance measurements of binding kinetics (Ka approximately 1x10(9) M-1). A competition ELISA using captured whole virus confirmed that the binding affinities of the parental scFv and also of hu7 and hu10 scFvs were identical. However, when compared with the original scFv, hu10 scFv was shown to have significantly decreased antiviral activity both in vitro and in a mouse model. Our observations suggest that binding of the scFv to the viral antigen is not sufficient for neutralization. We speculate that neutralization may involve the inhibition or induction of conformational changes in the bound antigen, thereby interfering with the F protein-mediated fusion of virus and cell membranes in the initial steps of infection.     

Computational and site-specific mutagenesis analyses of the asymmetric charge distribution on calmodulin

Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain alpha-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-dependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charge in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties of calmodulin are one of several properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.     

Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists

Background

Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance.

Results

Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G₄S)₄ were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent K_d values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells.

Conclusions

Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.     

Effects of PEGylated scFv antibodies against Plasmodium vivax duffy binding protein on the biological activity and stability in vitro

Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as dimers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity (KD=1.02 x 10(-7) M). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and play a critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.     

The Skp chaperone helps fold soluble proteins in vitro by inhibiting aggregation

The periplasmic seventeen kilodalton protein (Skp) chaperone has been characterized primarily for its role in outer membrane protein (OMP) biogenesis, during which the jellyfish-like trimeric protein encapsulates partially folded OMPs, protecting them from the aqueous environment until delivery to the BAM outer membrane protein insertion complex. However, Skp is increasingly recognized as a chaperone that also assists in folding soluble proteins in the bacterial periplasm. In this capacity, Skp coexpression increases the active yields of many recombinant proteins and bacterial virulence factors. Using a panel of single-chain antibodies and a single-chain T-cell receptor (collectively termed scFvs) possessing varying stabilities and biophysical characteristics, we performed in vivo expression and in vitro folding and aggregation assays in the presence or absence of Skp. For Skp-sensitive scFvs, the presence of Skp during in vitro refolding assays reduced aggregation but did not alter the observed folding rates, resulting in a higher overall yield of active protein. Of the proteins analyzed, Skp sensitivity in all assays correlated with the presence of folding intermediates, as observed with urea denaturation studies. These results are consistent with Skp acting as a holdase, sequestering partially folded intermediates and thereby preventing aggregation. Because not all soluble proteins are sensitive to Skp coexpression, we hypothesize that the presence of a long-lived protein folding intermediate renders a protein sensitive to Skp. Improved understanding of the bacterial periplasmic protein folding machinery may assist in high-level recombinant protein expression and may help identify novel approaches to block bacterial virulence.     

Investigation of the stability of yeast rad52 mutant proteins uncovers post-translational and transcriptional regulation of Rad52p

We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations--one a deletion of amino acids 210-327 and the other a missense mutation of residue 235--increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.     

Surface amino acids as sites of temperature-sensitive folding mutations in the P22 tailspike protein

Temperature-sensitive folding (tsf) mutations in the gene for the thermostable P22 tailspike interfere with the polypeptide chain folding and association pathway at restrictive temperature without altering the thermostability of the protein once correctly folded and assembled at permissive temperature. Though the native proteins matured at permissive temperature are biologically active, many of them display alterations in electrophoretic mobility. The native forms of 15 of these tsf mutant proteins have been purified and characterized. The purified proteins differed in electrophoretic mobility and isoelectric point from wild type but did not show evidence of major conformational alterations. The results suggest that the electrophoretic variations conferred by the 15 tsf amino acid substitutions are due to changes in the net charge at solvent-accessible sites in the native form of the mutant protein. During the maturation of the chains at restrictive temperature, these sites influence the conformation of intermediates in chain folding and association. The amino acid sequences at these sites resemble those found at turns in polypeptide chains. The isolation of tsf mutations requires that the mature structure of the tailspike accommodates the mutant amino acid substitution without loss of function. The solvent-accessible sites are probably at the surface of this structural protein. This would explain how bulky mutant substitutions, such as arginines for glycines, are accommodated in the native tailspike structure. Such sites, stabilizing intermediates in the folding pathway and located on the surface of the mature protein, probably represent a general class of conformational substrates for tsf mutations.     

Premature translation termination mediates triosephosphate isomerase mRNA degradation.

We characterized an anemia-inducing mutation in the human gene for triosephosphate isomerase (TPI) that resulted in the production of prematurely terminated protein and mRNA with a reduced cytoplasmic half-life. The mutation converted a CGA arginine codon to a TGA nonsense codon and generated a protein of 188 amino acids, instead of the usual 248 amino acids. To determine how mRNA primary structure and translation influence mRNA stability, in vitro-mutagenized TPI alleles were introduced into cultured L cells and analyzed for their effect on TPI RNA metabolism. Results indicated that mRNA stability is decreased by all nonsense and frameshift mutations. To determine the relative contribution of the changes in mRNA structure and translation to the altered half-life, the effects of individual mutations were compared with the effects of second-site reversions that restored translation termination to normal. All mutations that resulted in premature translation termination reduced the mRNA half-life solely or mainly by altering the length of the mRNA that was translated. The only mutation that altered translation termination and that reduced the mRNA half-life mainly by affecting the mRNA structure was an insertion that shifted termination to a position downstream of the normal stop codon.     

Kinetics of amyloid aggregation of mammal apomyoglobins and correlation with their amino acid sequences

In protein deposition disorders, a normally soluble protein is deposited as insoluble aggregates, referred to as amyloid. The intrinsic effects of specific mutations on the rates of protein aggregation and amyloid formation of unfolded polypeptide chains can be correlated with changes in hydrophobicity, propensity to convert alpha-helical to beta sheet conformation and charge. In this paper, we report the aggregation rates of buffalo, horse and bovine apomyoglobins. The experimental values were compared with the theoretical ones evaluated considering the amino acid differences among the sequences. Our results show that the mutations which play critical roles in the rate-determining step of apomyoglobin aggregation are those located within the N-terminal region of the molecule.     

Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.     

A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold.

We report the design, construction and use of an antibody bacteriophage display library built on the scaffold of a single-chain variable fragment (scFv) previously proven to be functionally expressed in the reducing environment of both bacterial and plant cytoplasm and endowed with intrinsic high thermodynamic stability. Four amino acid residues of the third hypervariable loop (CDR3) of both VH and VL were combinatorially mutated, generating a repertoire of approximately 5x10(7) independent scFvs, cloned in a phagemid vector. The ability of the antibody phage library to yield specific binders was tested by biopanning against several antigens. Successful selection of fully active scFvs was obtained, confirming the notion that combinatorial mutagenesis of few amino acid residues centrally located in the antigen-binding site is sufficient to provide binding specificities against virtually any target. High yields of both soluble and phage antibodies were obtained in Escherichia coli. Maintenance of the cognate scFv antibody stability in the newly selected scFv fragments was demonstrated by guanidinium chloride denaturation/renaturation studies and by soluble antibody expression in the bacterial cytoplasm. The antibody library described here allows the isolation of new stable binding specificities, potentially exploitable as immunochemical reagents for intracellular applications.     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

Importance of mutant position in Ramachandran plot for predicting protein stability of surface mutations

Understanding the mechanisms by which mutations affect protein stability is one of the most important problems in molecular biology. In this work, we analyzed the relationship between changes in protein stability caused by surface mutations and changes in 49 physicochemical, energetic, and conformational properties of amino acid residues. We found that the hydration entropy was the major contributor to the stability of surface mutations in helical segments; other properties responsible for size and volume of molecule also correlated significantly with stability. Classification of coil mutations based on their locations in the (phi-psi) map improved the correlation significantly, demonstrating the existence of a relationship between stability and strain energy, which indicates that the role of strain energy is very important for the stability of surface mutations. We observed that the inclusion of sequence and structural information raised the correlation, indicating the influence of surrounding residues on the stability of surface mutations. Further, we examined the previously reported "inverse relationship" between stability and hydrophobicity, and observed that the inverse hydrophobic effect was generally applicable only to coil mutations. The present study leads to a simple method for predicting protein stability changes caused by amino acid substitutions, which will be useful for protein engineering in designing novel proteins with increased stability and altered function.     

Stabilization of the cold shock protein CspB from Bacillus subtilis by evolutionary optimization of Coulombic interactions

The bacterial cold shock proteins (Csp) are used by both experimentalists and theoreticians as model systems for analyzing the Coulombic contributions to protein stability. We employ Proside, a method of directed evolution, to identify stabilized variants of Bs-CspB from Bacillus subtilis. Proside links the increased protease resistance of stabilized protein variants to the infectivity of a filamentous phage. Here, three cspB libraries were used for in vitro selections to explore the stabilizing potential of charged amino acids in Bs-CspB. In the first library codons for nine selected surface residues were partially randomized, in the second one random mutations were introduced non-specifically by error-prone PCR, and in the third one the spontaneous mutation rate of the phage in Escherichia coli was used. Stabilizing mutations were found at the surface positions 1, 3, 46, 48, 65, and 66. The contributions of these mutations to stability were characterized by analyzing them individually and in combination. The best combination (M1R, E3K, K65I, and E66L) increased the midpoint of thermal unfolding of Bs-CspB from 53.8 to 85.0 degrees C. The effects of most mutations are strongly context dependent. A good example is provided by the E3R mutation. It is strongly stabilizing (DeltaDeltaGD=11.1kJ mol(-1)) in the wild-type protein, but destabilizing (DeltaDeltaGD=-4.0kJ mol(-1)) in the A46K/S48R/E66L variant. The stabilizations by charge mutations did not correlate well with the corresponding changes in the protein net charge, and they could not be ascribed to the formation of ion pairs. Previous theoretical analyses did not identify the stabilization caused by the mutations at positions 1, 46, and 48. Also, electrostatics calculations based on protein net charge or charge asymmetry did not predict well the stability changes that occur when charged residues in Bs-CspB are mutated. It remains a challenge to model the Coulombic interactions of charged residues in a protein and to determine their contributions to the Gibbs free energy of protein folding.     

Functional analysis of predicted coiled-coil regions in the Escherichia coli K-12 O-antigen polysaccharide chain length determinant Wzz

Wzz is a membrane protein that determines the chain length distribution of the O-antigen lipopolysaccharide by an unknown mechanism. Wzz proteins consist of two transmembrane helices separated by a large periplasmic loop. The periplasmic loop of Escherichia coli K-12 Wzz (244 amino acids from K65 to A308) was purified and found to be a monomer with an extended conformation, as determined by gel filtration chromatography and analytical ultracentrifugation. Circular dichroism showed that the loop has a 60% helical content. The Wzz periplasmic loop also contains three regions with predicted coiled coils. To probe the function of the predicted coiled coils, we constructed amino acid replacement mutants of the E. coli K-12 Wzz protein, which were designed so that the coiled coils could be separate without compromising the helicity of the individual molecules. Mutations in one of the regions, spanning amino acids 108 to 130 (region I), were associated with a partial defect in O-antigen chain length distribution, while mutants with mutations in the region spanning amino acids 209 to 223 (region III) did not have an apparent functional defect. In contrast, mutations in the region spanning amino acids 153 to 173 (region II) eliminated the Wzz function. This phenotype was associated with protein instability, most likely due to conformational changes caused by the amino acid replacements, which was confirmed by limited trypsin proteolysis. Additional mutagenesis based on a three-dimensional model of region I demonstrated that the amino acids implicated in function are all located at the same face of a predicted α-helix, suggesting that a coiled coil actually does not exist in this region. Together, our results suggest that the regions predicted to be coiled coils are important for Wzz function because they maintain the native conformation of the protein, although the existence of coiled coils could not be demonstrated experimentally.     

Magnetizable antibody-like proteins

Use of paramagnetic particles to isolate molecules or cells from complex media is well established. Typically, particles are manufactured and coated with a biological molecule that confers specific biorecognition. Incubation of particles with sample and exposure to magnetic fields isolates the species of interest. We have designed, produced and assessed magnetized fusion proteins consisting of the antigen-binding portion of an antibody (single chain variable fraction; scFv) fused to the heavy chain of the iron-binding protein ferritin. The fusion protein subunits expressed in E. coli assemble to form a fusion protein consisting of a ferritin sphere with scFvs on the surface. The fusion proteins were chemically magnetized by introducing a paramagnetic iron core. The resultant fusion protein was shown to be magnetizable and capable of binding target antigens. These “organic” magnetizable particles possess a number of theoretical advantages over traditional inorganic particles.