Crystal structures of MBP fusion proteins

Although chaperone‐assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose‐binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter‐domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP‐mediated structural distortions are very rare.     

Genomic cloning,expression and recombinant protein purification of α and β subunits of the allophycocyanin gene <Emphasis Type="Italic">(apc)</Emphasis> from the cyanobacterium <Emphasis Type="BoldItalic">Anacystis nidulans</Emphasis> UTEX 625

A genomic fragment encoding α^APC and β^APC (i.e., α and β units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP (maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. α^APC and β^APC were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.     

Expression and activation of an esterase from Pseudomonas aeruginosa 1001 in Escherichia coli

An expression vector was constructed to overproduce a maltose binding protein (MBP)-esterase fusion protein in Escherichia coli. Soluble fusion protein was separated by centrifugation after cell disruption. The fusion protein was partially purified with amylose resin. The higher concentration of fusion protein (above 2 mg/ml) did not show any activity but about 0.3 mg/ml of fusion protein had the highest activity (142 U/ml). It is due to the difficulty of contact between substrate and active site of enzyme in compact form at high concentration. The fusion protein over-expressed could not be separated into MBP and esterase by the action of protease ‘Factor Xa’. The esterase could be cleaved from MBP fusion protein by the treatment of SDS with the Factor Xa, and the resulting esterase activity was increased to 34% after cleavage.     

Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins

It is difficult to imagine any strategy for high-throughput protein expression and purification that does not involve genetically engineered affinity tags. Because of its ability to enhance the solubility and promote the proper folding of its fusion partners, Escherichia coli maltose-binding protein (MBP) is a particularly useful affinity tag. However, not all MBP fusion proteins bind efficiently to amylose resin, and even when they do it is usually not possible to obtain a sample of adequate purity after a single affinity step. To address this problem, we endeavored to incorporate supplemental affinity tags within the framework of an MBP fusion protein. We show that both the nature of the supplemental tags and their location can influence the ability of MBP to promote the solubility of its fusion partners. The most promising configurations for high-throughput protein expression and purification appear to be a fusion protein with a biotin acceptor peptide (BAP) on the N-terminus of MBP and/or a hexahistidine tag (His-tag) on the C-terminus of the passenger protein. Abbreviatoins: BAP, biotin acceptor peptide; EDTA, ethelenediaminetetraacetic acid; IPTG, isopropyl--d-thiogalactopyranoside; MBP, E. coli maltose-binding protein; GFP; green fluorescent protein; Ni-NTA, nickel-nitrilotriacetic acid; ORF, open reading frame; PCR; polymerase chain reaction; R5, polyarginine tag; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEV, tobacco etch virus; WT, wild-type     

Expression and purification of recombinant proteins by fusion to maltose-binding protein

The pMAL vectors provide a method for purifying proteins from cloned genes by fusing them to maltose-binding protein (MBP, product of malE), which binds to amylose. The vectors use the tac promoter and the translation initiation signals of MBP to give high-level expression of the fusion, and an affinity purification for MBP to isolate the fusion protein. The pMAL polylinkers carry restriction sites to insert the gene of interest, and encode a site for a specific protease to separate MBP from the target protein after purification. Vectors with or without the malE signal sequence can be used, to express the protein cytoplasmically for the highest level of production or periplasmically to help in proper folding of disulfide-bonded proteins.     

Solubility-enhancing proteins MBP and NusA play a passive role in the folding of their fusion partners

It is well established that certain highly soluble proteins have the ability to enhance the solubility of their fusion partners. However, very little is known about how different solubility enhancers compare in terms of their ability to promote the proper folding of their passenger proteins. We compared the ability of two well-known solubility enhancers, Escherichia coli maltose-binding protein (MBP) and N utilization substance A (NusA), to improve the solubility and promote the proper folding of a variety of passenger proteins that are difficult to solubilize. We used an intracellular processing system to monitor the solubility of these passenger proteins after they were cleaved from MBP and NusA by tobacco etch virus protease. In addition, the biological activity of some fusion proteins was compared to serve as a more quantitative indicator of native structure. The results indicate that MBP and NusA have comparable solubility-enhancing properties. Little or no difference was observed either in the solubility of passenger proteins after intracellular processing of the MBP and NusA fusion proteins or in the biological activity of solubilized passenger proteins, suggesting that the underlying mechanism of solubility enhancement is likely to be similar for both the proteins, and that they play a passive role rather than an active one in the folding of their fusion partners.     

Production of Bioactive Rockfish (<Emphasis Type="Italic">Sebastes schlegeli</Emphasis>) Myostatin-1 Prodomain in an <Emphasis Type="Italic">Escherichia coli</Emphasis> System

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth in mammalian species, and its activity is inhibited by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing. In fish, MSTN also appears to suppress fish muscle growth with its activity being inhibited by prodomain. The objective of this study was to produce bioactive MSTN-1 prodomain of rockfish (S. schlegeli), a commercial aquaculture species in East Asia, in E. coli using maltose binding protein (MBP) as a fusion partner. Rockfish MSTN-1 prodomain (sMSTN1pro) cDNA was cloned into the pMALc2x vector, and proteins (MBP-sMSTN1pro) were expressed in Rosetta-gami 2(DE3)pLysS cells by IPTG induction. The MBP-sMSTN1pro was expressed in soluble forms, and affinity purified using amylose resin. The affinity purified MBP-sMSTN1pro suppressed MSTN activity in vitro. The results suggest that MBP is probably a useful fusion partner in producing bioactive MSTN prodomains of various animal species in E. coli.     

A dual protease approach for expression and affinity purification of recombinant proteins

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His₆-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His₆-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His₆ tag is removed by His₆-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His₆-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification.     

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

Myelin basic protein (MBP) is a major protein of myelin‐proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12‐mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti‐MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (K _d = 0.51–0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10⁻¹ to 2.3 × 10⁻⁴ M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (K _d, M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192‐fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins.     

Overproduction and purification of Lon protease fromEscherichia coli using a maltose-binding protein fusion system

Lon protease, which plays a major role in degradation of abnormal proteins inEscherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form inE. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl--d-methoxynaphthylamide) and protein (-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes.     

Purification and in vitro characterization of the maltose-binding protein of the plant pathogen Xanthomonas citri

The uptake of maltose and maltodextrins in gram-negative bacteria is mediated by an ATP-dependent transport complex composed of a periplasmic maltose-binding protein (MBP) and membrane-associated proteins responsible for the formation of a membrane pore and generation of energy to drive the translocation process. In this work, we report the purification and in vitro functional analysis of MBP, encoded by the malE gene, of the plant pathogen Xanthomonas citri, responsible for the canker disease affecting citrus plants throughout the world. The X. citri MBP is composed of 456 amino acids, displaying a low amino acid identity (16% throughout the sequence) compared to the Escherichia coli K12 ortholog. The X. citri malE gene was cloned into a pET28a vector, and the encoded protein was expressed and purified by affinity chromatography as a His-tag N-terminal fusion peptide produced by the E. coli BL21 strain. Enhanced levels of soluble protein were achieved with static cultures kept overnight at 23 degrees C. Ability to bind immobilized amylose, the emission of intrinsic fluorescence and circular dichroism spectra indicated that the purified recombinant protein preserved both conformation and biological activity of the native protein. The availability of the recombinant MBP will contribute to the functional and structural analysis of the maltose and maltodextrin uptake system of the plant pathogen X. citri.     

High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin

To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.     

Abeta produced as a fusion to maltose binding protein can be readily purified and stably associates with copper and zinc

The 42 amino acid Alzheimer's Abeta peptide has been produced in E. coli as a soluble fusion to maltose binding protein (MBP). Affinity purification on amylose columns of MBP-Abeta and MBP led to the recovery of proteins at purities that were suited for physicochemical analyses. MBP-Abeta was able to bind approximately 2 mole equivalents of copper or 4 mole equivalents of zinc, while MBP alone bound negligible amounts of zinc or copper. We conclude that Abeta can bind 2 copper or 4 zinc ions in its fusion format. Because MBP-Abeta is a convenient protein to work with, this system is well suited for further studies on the structure of Abeta and its interactions with metals.     

An ISFET biosensor for the monitoring of maltose-induced conformational changes in MBP

Here we describe an ion sensitive field effect transistor (ISFET) biosensor, which was designed to monitor directly the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. This study is the first report of the application of a FET biosensor to the monitoring of conformationally changed proteins. Consequently, a significant drop in current on the basis of the charge-dependent capacitance measurement has been clearly observed in response to maltose, but not for the glucose control, thereby indicating that the substrate-specific conformational properties of the target protein could be successfully monitored using the ISFET. Collectively, our results clearly suggest that ISFET provide a high fidelity system for the detection of maltose-induced structural alterations in MBP.     

Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.     

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)⁺ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP maltose binding protein - PCR polymerase chain reaction - Stress70c the cytosolic member of the 70 kDA family of stress-related proteins     

Thermodynamics of maltose binding protein unfolding.

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a delta(Hcal)/delta(HvH) ratio in the range of 1.3-1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the Tm by 8-15 degrees C, depending of pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The delta(Hcal)/delta(HvH) ratio decreased to unity, indicating that the two domains melt together in a single two-state transition. This ligand-induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the delta(Hcal)/delta(HvH) ratio. Binding of maltose to MBP is characterized by very low enthalpy changes, approximately -1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity. 0.16 cal/K-g, which is consistent with the high amount of nonpolar surface--0.72 A2/g--that becomes accessible to solvent in the unfolded state. The high value of delta Cp determines a very steep delta G versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5 degrees C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.     

抗MRP3单链抗体-sTRAIL融合蛋白诱导U251多形性胶质母细胞瘤凋亡

采用重组PCR技术获得抗多药耐药相关蛋白3(multidrug resistance protein 3, MRP3)的单链抗体(scFv)与人源可溶性肿瘤坏死因子相关凋亡诱导配体(soluble TNF-related apoptosis inducing ligand, sTRAIL)的融合蛋白质的基因编码序列, 利用原核表达载体pMAL-c2,构建含麦芽糖结合蛋白(maltose binding protein, MBP)标签肽的antiMRP3(scFv)-sTRAIL融合蛋白, 经亲和层析柱纯化. 获得纯化的antiMRP3(scFv)-sTRAIL融合蛋白,用MRP3阳性U251多形性胶质母细胞瘤做增殖抑制实验、细胞凋亡诱导实验,结果均显示具有明显的活性, 而MBP无明显作用. 上述结果表明,成功表达了antiMRP3(scFv)-sTRAIL融合蛋白, 该融合蛋白具有诱导U251多形性胶质母细胞瘤细胞凋亡的活性, 为开发靶向性抗肿瘤药物奠定了基础.     

The soluble expression of the human renin binding protein using fusion partners: A comparison of ubiquitin,thioredoxin, maltose binding protein and NusA

human renin binding protein (hRnBp), showingN-acetylglucosamine-2-epimerase activity, was over-expressed inE. coli, but was mainly present as an inclusion body. To improve its solubility and activity, ubiquitin (Ub), thioredoxin (Trx), maltose binding protein (MBP) and NusA, were used as fusion partners. The comparative solubilities of the fusion proteins were, from most to least soluble: NusA, MBP, Trx, Ub. Only the MBP fusion did not significantly reduce the activity of hRnBp, but enhanced the stability. The Origami (DE3), permitting a more oxidative environment for the cytoplasm inE. coli, helped to increase its functional activity.     

Detection of conformationally changed MBP using intramolecular FRET

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E) = ∼0.11, Distance (D) = ∼6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.