Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

Expression of scFv‐Mel‐Gal4 triple fusion protein as a targeted DNA‐carrier in Escherichia Coli

Liver‐directed gene therapy has become a promising treatment for many liver diseases. In this study, we constructed a multi‐functional targeting molecule, which maintains targeting, endosome‐escaping, and DNA‐binding abilities for gene delivery. Two single oligonucleotide chains of Melittin (M) were synthesized. The full‐length cDNA encoding anti‐hepatic asialoglycoprotein receptor scFv C1 (C1) was purified from C1/pIT2. The GAL4 (G) gene was amplified from pSW50‐Gal4 by polymerase chain reaction. M, C1 and G were inserted into plasmid pGC4C26H to product the recombinant plasmid pGC‐C1MG. The fused gene C1MG was subsequently subcloned into plasmid pET32c to product the recombinant plasmid C1MG/pET32c and expressed in Escherichia coli BL21. The scFv‐Mel‐Gal4 triple fusion protein (C1MG) was purified with a Ni²⁺ chelating HiTrap HP column. The fusion protein C1MG of roughly 64 kD was expressed in inclusion bodies; 4.5 mg/ml C1MG was prepared with Ni²⁺ column purification. Western blot and immunohistochemistry showed the antigen‐binding ability of C1MG to the cell surface of the liver‐derived cell line and liver tissue slices. Hemolysis testing showed that C1MG maintained membrane‐disrupting activity. DNA‐binding capacity was substantiated by luciferase assay, suggesting that C1MG could deliver the DNA into cells efficiently on the basis of C1MG. Successful expression of C1MG was achieved in E. coli, and C1MG recombinant protein confers targeting, endosome‐escaping and DNA‐binding capacity, which makes it probable to further study its liver‐specific DNA delivery efficacy in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural and functional characterization of an anti‐West Nile virus monoclonal antibody and its single‐chain variant produced in glycoengineered plants

Previously, our group engineered a plant‐derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single‐chain variable fragment (scFv) fused to the heavy chain constant domains (C_H) of human IgG (pE16scFv‐C_H). pE16 and pE16scFv‐C_H were expressed and assembled efficiently in Nicotiana benthamiana ?XF plants, a glycosylation mutant lacking plant‐specific N‐glycan residues. Glycan analysis revealed that ?XF plant‐derived pE16scFv‐C_H (?XFpE16scFv‐C_H) and pE16 (?XFpE16) both displayed a mammalian glycosylation profile. ?XFpE16 and ?XFpE16scFv‐C_H demonstrated equivalent antigen‐binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell‐produced E16 (mE16). A single dose of ?XFpE16 or ?XFpE16scFv‐C_H protected mice against WNV‐induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single‐chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti‐WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N‐glycosylation. This platform may lead to a more robust and cost‐effective production of antibody‐based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.     

c‐Met function requires n‐linked glycosylation modification of pro‐Met

c‐Met, the receptor for hepatocyte growth factor (HGF), is cell surface tyrosine kinase that controls cancer cell growth, survival, invasion, and metastasis. Post‐translational modification, such as glycosylation, plays an essential role in regulating the function of cell surface molecules. Whether glycosylation modification regulates the enzymatic properties of c‐Met is unknown. In this study, we investigated the effect of glycosylation on the function of c‐Met. We found that c‐Met is an N‐linked glycosylated protein. Both pro‐Met and p145Met (the β subunit of mature c‐Met) have N‐linked glycosylation. Glycosylation inhibitor studies revealed that the N‐glycosylation modification of p145Met is from pro‐Met, but not due to the further modification of pro‐Met. Importantly, blocking the N‐glycosylation targets pro‐Met to cytoplasm and initiates its phosphorylation independent of HGF engagement. Nonglycosylated pro‐Met activates c‐Met downstream pathways to a certain extent to compensate for the degradation of p145Met induced by glycosylation blocking‐mediated endoplasmic reticulum (ER) stress. J. Cell. Biochem. 114: 816–822, 2013. © 2012 Wiley Periodicals, Inc.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Establishing microbial co‐cultures for 3‐hydroxybenzoic acid biosynthesis on glycerol

Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designed E. coli–E. coli co‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E. coli mono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis.     

Functional expression of anti-hepatitis B virus (HBV) preS2 antigen scFv by <Emphasis Type="Italic">cspA</Emphasis> promoter system in <Emphasis Type="Italic">Escherichia coli</Emphasis> and application as a recognition molecule for single-walled carbon nanotube (SWNT) field effect transistor (FET)

The preS2 antigens of hepatitis B virus (HBV), which causes a serious health problem in the world, have been implicated in hepatocyte cell binding and viral penetration. Therefore, the importance of antibody production against preS2 antigen for early diagnosis of HBV has been well established. In this study, the recombinant HBV preS2 single chain variable fragment (scFv) antibody was successfully expressed in E. coli with the novel cold shock vector (pCold) under the cspA promoter, and its expression level was compared with the pET vector under the T7 promoter. Additionally, a host with an oxidizing cytoplasm, E. coli trxB/gor double mutant, was used to improve the soluble expression. The anti-HBV preS2 scFv using pCold vector was successfully expressed in a soluble and functional form in both wild type and double mutant E. coli, while the scFv using the pET vector was expressed in an insoluble form in spite of using a double mutant providing an oxidizing environment. The induction with 0.05 mM IPTG showed a 2-fold higher functional expression compared to induction with 1 mM IPTG, and the functional expression at the induction temperature (15°C), which is optimal temperature for pCold vector, was improved 2-fold and 3- fold at 4 and 25°C, respectively. The efficacy of anti-HBV preS2 scFv for detecting HBV preS2 antigen was tested and verified by using Ni-decorated single-walled carbon nanotube (SWNT) field effect transistors.     

On the mechanism of protein fold‐switching by a molecular sensor

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild‐type fold (N) to a circularly‐permuted fold (N′), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order–disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D_9k. A simple three‐state model for fold switching holds that N and N′ interconvert through the unfolded state. This model predicts that the fundamental properties of the switch—calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate—can be controlled by altering the relative stabilities of N and N′. We find that selectively destabilizing N or N′ changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole‐molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Effects of variable domain orientation on anti-HER2 single-chain variable fragment antibody expressed in the Escherichia coli cytoplasm

Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.     

Improved antimicrobial activity of h‐lysozyme (107–115) by rational Ala substitution

The most challenging target in the design of new antimicrobial agents is the development of antibiotic resistance. Antimicrobial peptides are good candidates as lead compounds for the development of novel anti‐infective drugs. Here we propose the sequential substitution of each Ala residue present in a lead peptide with known antimicrobial activity by specific amino acids, rationally chosen, that could enhance the activity of the resultant peptide. Taking the fragment 107–115 of the human lysozyme as lead, two‐round screening by sequentially replacing both Ala residues (108 and 111) by distinct amino acids resulted in a novel peptide with 4‐ and 20‐fold increased antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. These results reinforce the strategy proposed, which, in combination with simple and easy screening tools, will contribute to the rapid development of new therapeutic peptides required by the market. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering of Phosphoserine Aminotransferase Increases the Conversion of l‐Homoserine to 4‐Hydroxy‐2‐ketobutyrate in a Glycerol‐Independent Pathway of 1,3‐Propanediol Production from Glucose

Phosphoserine aminotransferase (SerC) from Escherichia coli (E. coli) MG1655 is engineered to catalyze the deamination of homoserine to 4‐hydroxy‐2‐ketobutyrate, a key reaction in producing 1,3‐propanediol (1,3‐PDO) from glucose in a novel glycerol‐independent metabolic pathway. To this end, a computation‐based rational approach is used to change the substrate specificity of SerC from l ‐phosphoserine to l ‐homoserine. In this approach, molecular dynamics simulations and virtual screening are combined to predict mutation sites. The enzyme activity of the best mutant, SerC_R42W/R77W, is successfully improved by 4.2‐fold in comparison to the wild type when l ‐homoserine is used as the substrate, while its activity toward the natural substrate l ‐phosphoserine is completely deactivated. To validate the effects of the mutant on 1,3‐PDO production, the “homoserine to 1,3‐PDO” pathway is constructed in E. coli by coexpression of SerC_R42W/R77W with pyruvate decarboxylase and alcohol dehydrogenase. The resulting mutant strain achieves the production of 3.03 g L^?1 1,3‐PDO in fed‐batch fermentation, which is 13‐fold higher than the wild‐type strain and represents an important step forward to realize the promise of the glycerol‐independent synthetic pathway for 1,3‐PDO production from glucose.     

Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full‐size antibody in BY‐2 suspension culture

Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture‐produced proteins such as full‐size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram‐per‐liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full‐size human anti‐HIV antibody 2G12 in N. tabacum cv. BY‐2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10–20‐fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred‐tank bioreactors, where the maximum yield per cultivation volume was 8 mg L^?1 over 7 days. During the stationary phase, antibody levels were 150‐fold higher in nitrogen‐enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N‐glycan analysis. Biotechnol. Bioeng. 2010;107: 278–289. © 2010 Wiley Periodicals, Inc.     

Coexpression of molecular chaperones to enhance functional expression of anti-BNP scFv in the cytoplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the detection of B-type natriuretic peptide

Molecular chaperones are a ubiquitous family of cellular proteins that mediate the correct folding of other target polypeptides. In our previous study, the recombinant anti-BNP scFv, which has promising applications for diagnostic, prognostic, and therapeutic monitoring of heart failure, was expressed in the cytoplasm of Escherichia coli. However, when the anti-BNP scFv was expressed, 73.4% of expressed antibodies formed insoluble inclusion bodies. In this study, molecular chaperones were coexpressed with anti-BNP scFv with the goal of improving the production of functional anti-BNP in the cytoplasm of E. coli. Five sets of molecular chaperones were assessed for their effects on the production of active anti-BNP scFv. These sets included the following: trigger factor (TF); groES/groEL; groES/groEL/TF; dnaK/dnaJ/grpE; groES/groEL/dnaK/dnaJ/grpE. Of these chaperones, the coexpression of anti-BNP scFv with the groES/groEL chaperones encoded in plasmid pGro7 exhibited the most efficient functional expression of anti-BNP scFv as an active form. Coexpressed with the groES/groEL chaperones, 64.9% of the total anti-BNP scFv was produced in soluble form, which is 2.4 times higher scFv than that of anti-BNP scFv expressed without molecular chaperones, and the relative binding activity was 1.5-fold higher. The optimal concentration of l-arabinose required for induction of the groES/groEL chaperone set was determined to be 1.0 mM and relative binding activity was 3.5 times higher compared with that of no induction with l-arabinose. In addition, soluble anti-BNP scFv was increased from 11.5 to 31.4 μg/ml with optimized inducer concentration (1.0 mM l-arabinose) for the coexpression of the groES/groEL chaperones. These results demonstrate that the functional expression of anti-BNP scFv can be improved by coexpression of molecular chaperones, as molecular chaperones can identify and help to refold improperly folded anti-BNP scFv.     

Improving GPX activity of selenium‐containing human single‐chain Fv antibody by site‐directed mutation based on the structural analysis

Glutathione peroxidase (GPX) is one of the important members of the antioxidant enzyme family. It can catalyze the reduction of hydroperoxides with glutathione to protect cells against oxidative damage. In previous studies, we have prepared the human catalytic antibody Se‐scFv‐B3 (selenium‐containing single‐chain Fv fragment of clone B3) with GPX activity by incorporating a catalytic group Sec (selenocysteine) into the binding site using chemical mutation; however, its activity was not very satisfying. In order to try to improve its GPX activity, structural analysis of the scFv‐B3 was carried out. A three‐dimensional (3D) structure of scFv‐B3 was constructed by means of homology modeling and binding site analysis was carried out. Computer‐aided docking and energy minimization (EM) calculations of the antibody‐GSH (glutathione) complex were also performed. From these simulations, Ala44 and Ala180 in the candidate binding sites were chosen to be mutated to serines respectively, which can be subsequently converted into the catalytic Sec group. The two mutated protein and wild type of the scFv were all expressed in soluble form in Escherichia coli Rosetta and purified by Ni²⁺‐immobilized metal affinity chromatography (IMAC), then transformed to selenium‐containing catalytic antibody with GPX activity by chemical modification of the reactive serine residues. The GPX activity of the mutated catalytic antibody Se‐scFv‐B3‐A180S was significantly increased compared to the original Se‐scFv‐B3. Copyright © 2009 John Wiley & Sons, Ltd.     

Accurate prediction of protein folding rates from sequence and sequence‐derived residue flexibility and solvent accessibility

Protein folding rates vary by several orders of magnitude and they depend on the topology of the fold and the size and composition of the sequence. Although recent works show that the rates can be predicted from the sequence, allowing for high‐throughput annotations, they consider only the sequence and its predicted secondary structure. We propose a novel sequence‐based predictor, PFR‐AF, which utilizes solvent accessibility and residue flexibility predicted from the sequence, to improve predictions and provide insights into the folding process. The predictor includes three linear regressions for proteins with two‐state, multistate, and unknown (mixed‐state) folding kinetics. PFR‐AF on average outperforms current methods when tested on three datasets. The proposed approach provides high‐quality predictions in the absence of similarity between the predicted and the training sequences. The PFR‐AF's predictions are characterized by high (between 0.71 and 0.95, depending on the dataset) correlation and the lowest (between 0.75 and 0.9) mean absolute errors with respect to the experimental rates, as measured using out‐of‐sample tests. Our models reveal that for the two‐state chains inclusion of solvent‐exposed Ala may accelerate the folding, while increased content of Ile may reduce the folding speed. We also demonstrate that increased flexibility of coils facilitates faster folding and that proteins with larger content of solvent‐exposed strands may fold at a slower pace. The increased flexibility of the solvent‐exposed residues is shown to elongate folding, which also holds, with a lower correlation, for buried residues. Two case studies are included to support our findings. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs

B. licheniformis exo‐small β‐lactamase (ESBL) has a complex architecture with twelve α helices and a five‐stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL α helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.