Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC₅₀ values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.     

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor β (rh TNF-β) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rh TNF-β from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm^?3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.     

Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.     

Refolded scFv Antibody Fragment against Myoglobin Shows Rapid Reaction Kinetics

Myoglobin is one of the early biomarkers for acute myocardial infarction. Recently, we have screened an antibody with unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid reaction kinetics are thought to be an early IgG form produced during early stage of in vivo immunization. We produced a recombinant scFv fragment for the premature antibody from Escherichia coli using refolding technology. The scFv gene was constructed by connection of the V_H–V_L sequence with a (Gly₄Ser)₃ linker. The scFv fragment without the pelB leader sequence was expressed at a high level, but the solubility was extremely low. A high concentration of 8 M urea was used for denaturation. The dilution refolding process in the presence of arginine and the redox reagents GSH and GSSH successfully produced a soluble scFv protein. The resultant refolded scFv protein showed association and dissociation values of 9.32 × 10⁻⁴ M⁻¹·s⁻¹ and 6.29 × 10⁻³ s⁻¹, respectively, with an affinity value exceeding 10⁷ M⁻¹ (k_on/k_off), maintaining the original rapid reaction kinetics of the premature antibody. The refolded scFv could provide a platform for protein engineering for the clinical application for diagnosis of heart disease and the development of a continuous biosensor.     

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption

Engineering of biomimetic motives have emerged as promising approaches to improving cells’ binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface’s receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5β1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.     

Rational design of `water-soluble' bacteriorhodopsin variants

We have explored the interchangeability of soluble and membraneproteins by attempting to render a helical membrane protein`water soluble' through mutation of its lipid-exposed residues.Using an atomic resolution structure of bacteriorhodopsin (bR),two different strategies were developed to identify lipid-exposedresidues for mutation. In the first strategy all residues intrimeric bR with solvent accessibility >35% were marked forreplacement. Replacement residues were chosen so as to map anaverage surface of helical soluble proteins onto the bR surface,resulting in the mutagenesis of 14.9% of surface residues. Thesecond strategy took into account the observation that accessibleresidues can be categorized as fully or partially accessible.Consequently, three mutants were designed based on monomericbR, all with their accessible residues changed and with varyingextents of mutagenesis of partially accessible residues. 13.5–24.3%of the wild-type surface was altered in these designs. The constructfor the first design was cloned into Escherichia coli. Traceamounts of the mutant protein were expressed with the concurrentoverexpression of an endogenous prolyl isomerase. In contrast,all three mutant proteins of the second design expressed welland could be purified to homogeneity. Systematic refolding trialswere undertaken with limited success at solubilization in aqueousmedia. We have discussed the feasibility of applying the `solubilizationstrategy' outlined here to membrane proteins.     

Box-Behnken设计法优化大肠杆菌重组DsBA蛋自纯化工艺(英文)

Disulfide bond formation protein A (DsbA) is one of the important helper proteins for folding in protein synthesis in vivo. In this study, purification of recombinant DsbA was investigated by examining four important factors with Box-Behnken design method, a statistic-based design of experiments. The optimal operation conditions were obtained by adopting the effectiveness coefficient method on the multi-objective problem, which takes the protein recovery, purification efficiency and throughput of ion-exchange chromatography into account. After the optimization, protein recovery of 96.8% and purity higher than 95% DsbA was achieved, and the productivity was (377.9±1.7)mg soluble DsbA per liter broth. The purified protein was identified by peptide mass fingerprinting matching the record of gi|2624856, a mutant of DsbA. The DsbA was preliminarily applied to the refolding of denatured lysozyme in vitro.     

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.     

Green Fluorescent Protein (GFP)-Based Overexpression Screening and Characterization of AgrC,a Receptor Protein of Quorum Sensing in Staphylococcus aureus

Staphylococcus aureus AgrC is an important component of the agr quorum-sensing system. AgrC is a membrane-embedded histidine kinase that is thought to act as a sensor for the recognition of environmental signals and the transduction of signals into the cytoplasm. However, the difficulty of expressing and purifying functional membrane proteins has drastically hindered in-depth understanding of the molecular structures and physiological functions of these proteins. Here, we describe the high-yield expression and purification of AgrC, and analyze its kinase activity. A C-terminal green fluorescent protein (GFP) fusion to AgrC served as a reporter for monitoring protein expression levels in real time. Protein expression levels were analyzed by the microscopic assessment of the whole-cell fluorescence. The expressed AgrC-GFP protein with a C-terminal His-tagged was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) at yields of ≥10 mg/L, following optimization. We also assessed the effects of different detergents on membrane solubilization and AgrC kinase activity, and polyoxyethylene-(23)-lauryl-ether (Brij-35) was identified as the most suitable detergent. Furthermore, the secondary structural stability of purified AgrC was analyzed using circular dichroism (CD) spectroscopy. This study may serve as a general guide for improving the yields of other membrane protein preparations and selecting the appropriate detergent to stabilize membrane proteins for biophysical and biochemical analyses.     

Alpha-Helical Protein KfrC Acts as a Switch between the Lateral and Vertical Modes of Dissemination of Broad-Host-Range RA3 Plasmid from IncU (IncP-6) Incompatibility Group

KfrC proteins are encoded by the conjugative broad-host-range plasmids that also encode alpha-helical filament-forming KfrA proteins as exemplified by the RA3 plasmid from the IncU incompatibility group. The RA3 variants impaired in kfrA, kfrC, or both affected the host’s growth and demonstrated the altered stability in a species-specific manner. In a search for partners of the alpha-helical KfrC protein, the host’s membrane proteins and four RA3-encoded proteins were found, including the filamentous KfrA protein, segrosome protein KorB, and the T4SS proteins, the coupling protein VirD4 and ATPase VirB4. The C-terminal, 112-residue dimerization domain of KfrC was involved in the interactions with KorB, the master player of the active partition, and VirD4, a key component of the conjugative transfer process. In Pseudomonas putida, but not in Escherichia coli, the lack of KfrC decreased the stability but improved the transfer ability. We showed that KfrC and KfrA were involved in the plasmid maintenance and conjugative transfer and that KfrC may play a species-dependent role of a switch between vertical and horizontal modes of RA3 spreading.     

Adenovirus Terminal Protein Contains a Bipartite Nuclear Localisation Signal Essential for Its Import into the Nucleus

Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5′end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP–host interactions further, we expressed TP in Escherichia coli (E. coli). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.     

Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle,Epicauta chinensis

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.     

Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1–R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment’s propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli.     

Recent advances in biomolecular process intensification

Escherichia coli has been the most popular cellular biofactory for the production of many biomolecules including recombinant proteins whose bioactivities are not dependent on complicated post-translational modifications such as glycosylation. Despite many advantages of protein production using E. coli, the formation of insoluble inclusion bodies (IBs) poses a major challenge to harnessing E. coli as a host of choice. Furthermore, the complicated processing steps associated with IB extraction, solubilisation of IBs and refolding of the solubilised-denatured IB proteins, which are typically encountered in the traditional IB processing flowsheet, significantly compromise process economics of IB-route protein production using E. coli. In this paper, many recent advances in innovative IB processing technologies are reviewed with a special emphasis on their potential to contribute to process intensification, which is critical to achieve better process economics in the IB-route recombinant protein production using E. coli.     

Increased Systemic Antioxidant Power Ameliorates the Aging-Related Reduction in Oocyte Competence in Mice

Ovarian aging is associated with elevated oxidative stress and diminished oocyte developmental competence. We aimed to determine the impact of systemic antioxidant treatment in aged mice. Female outbred CF-1 mice were aged for 9 months prior to an 8-week 45 mg Euterpe oleracea (açaí) daily supplement. The açaí treatment induced a threefold increase in serum antioxidant power (FRAP) compared to both young and aged mice (p < 0.0001). Compared to young mice, aged mice had fewer oocytes and reduced blastocyst development (p < 0.0001); açaí did not affect the oocyte numbers, but improved blastocyst formation (p < 0.05). Additionally, açaí alleviated the aging-related decrease in implantation potential (p < 0.01). The aged mice showed evidence of elevated ovarian ER stress (increased whole-ovary PDIA4 expression, granulosa cell and oocyte GRP78 expression, and oocyte PDIA4 protein), reduced oocyte mitochondrial quality (higher PRKN activation and mitochondrial DNA oxidative damage), and dysregulated uterine glandular epithelium. Antioxidant intervention was sufficient to lessen these effects of ovarian aging, likely in part by the upregulation of NRF2. We conclude that açaí treatment is a promising strategy to improve ER and mitochondrial function in the ovaries, thereby ameliorating the decreased oocyte competence that occurs with ovarian aging.     

表面活性剂辅助溶菌酶复性:表面活性剂-蛋白质复合物结构分析

研究了十六烷基三甲基溴化铵（CTAB）辅助天然溶菌酶的复性过程及其影响因素.发现向复性液中单独添加低浓度CTAB即可导致溶菌酶复性,且当变性溶菌酶浓度在0.2 mg&#8226;ml^-1以上、CTAB与溶菌酶的摩尔比为10时复性效果最佳.采用表面张力、离子交换色谱、非还原性SDS-PAGE及荧光发射光谱法对复性体系进行物性和结构分析,显示CTAB可与变性溶菌酶形成不同结构的表面活性剂-蛋白质复合物,且复合物的组成和结构可随时间发生变化.最后,对于表面活性剂-蛋白质复合物形成过程机理进行了探讨.     

Identification of Apolipoprotein C-I as a Potential Wilms’ Tumor Marker after Excluding Inflammatory Factors

Wilms’ tumor is one of the most common malignant tumors observed in children, and its early diagnosis is important for late-stage treatment and prognosis. We previously screened and identified protein markers for Wilms’ tumor; however, these markers lacked specificity, and some were associated with inflammation. In the current study, serum samples from children with Wilms’ tumors were compared with those of healthy controls and patients with systemic inflammatory response syndrome (SIRS). After exclusion of factors associated with inflammation, specific protein markers for Wilms’ tumors were identified. After comparing the protein peak values obtained from all three groups, a protein with a m/z of 6438 Da was specified. Purification and identification of the target protein using high-pressure liquid chromatography (HPLC) and two-dimensional liquid chromatography-linearion trap mass spectrometry(2D-LC-LTQ-MS) mass spectrometry, respectively, revealed that it was apolipoprotein C-I (APO C-I). Thus, APO C-I is a specific protein marker for Wilms’ tumor.     

Participation of Amyloid and Tau Protein in Post-Ischemic Neurodegeneration of the Hippocampus of a Nature Identical to Alzheimer’s Disease

Recent evidence suggests that amyloid and tau protein are of vital importance in post-ischemic death of CA1 pyramidal neurons of the hippocampus. In this review, we summarize protein alterations associated with Alzheimer’s disease and their gene expression (amyloid protein precursor and tau protein) after cerebral ischemia, as well as their roles in post-ischemic hippocampus neurodegeneration. In recent years, multiple studies aimed to elucidate the post-ischemic processes in the development of hippocampus neurodegeneration. Their findings have revealed the dysregulation of genes for amyloid protein precursor, β-secretase, presenilin 1 and 2, tau protein, autophagy, mitophagy, and apoptosis identical in nature to Alzheimer’s disease. Herein, we present the latest data showing that amyloid and tau protein associated with Alzheimer’s disease and their genes play a key role in post-ischemic neurodegeneration of the hippocampus with subsequent development of dementia. Therefore, understanding the underlying process for the development of post-ischemic CA1 area neurodegeneration in the hippocampus in conjunction with Alzheimer’s disease-related proteins and genes will provide the most important therapeutic development goals to date.