Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1α (stromal cell-derived factor-1α) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1α fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP–RANTES and MBP–SDF-1α was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1α.     

Expression, purification, and antibody binding activity of human papillomavirus 16 L1 protein fused to maltose binding protein

Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.     

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.     

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.     

Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli.

The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal 'solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression.     

The extracellular domain of the human erythropoietin receptor: expression as a fusion protein in Escherichia coli, purification, and biological properties

We developed an efficient production system of the soluble extracellular domain of the human erythropoietin receptor (sEPO-R) and characterized the binding of erythropoietin (EPO) with the purified recombinant protein. The sEPO-R, fused to the maltose binding protein (MBP), was expressed as a soluble protein in the periplasm of Escherichia coli (E. coli) and did not accumulate in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose followed by size exclusion chromatography (SEC). Specific binding of 125I-labelled EPO to the sEPO-R was demonstrated by competitive and saturation binding assays. A single affinity class (Kd = 0.25 nM) of the binding site was evident by Scatchard analysis. This value is similar to the Kd observed between EPO and the EPO-R of high affinity present on human erythroid progenitors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.     

Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis.

We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 degrees C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein.     

Single-step purification of proteins of interest from proteolytically cleaved recombinant maltose-binding protein (MBP) fusion proteins by selective immunoprecipitation of MBP

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins inE. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated by centrifugation, resulting in highly purified proteins in the supernatant.     

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.     

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.     

Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system.

Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK2), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK2 complex and that MBP plays a crucial role in triggering the transport process.     

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.     

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.     

不同蛋白标签对LMO2融合蛋白沉淀实验的影响

融合蛋白沉淀技术是一种用来研究蛋白质相互作用的新的体外实验技术, 通常利用蛋白亲和标签与探针蛋白融合表达来钓取未知相互作用蛋白或验证已知蛋白间的相互作用, 其中以谷胱甘肽巯基转移酶(GST)标签最为常用。LMO2(由LIM only缩写得名, 也称Ttg-2或Rbtn2)是一种小分子量难溶蛋白。利用原核系统分别表达了含有GST和麦芽糖结合蛋白(MBP)两种标签的LMO2融合蛋白, 发现GST-LMO2融合蛋白以包涵体的形式表达, 而MBP-LMO2融合蛋白则能够以可溶形式表达, 而且MBP-LMO2的表达量明显高于GST-LMO2融合蛋白。将可溶性的MBP-LMO2融合蛋白和复性后的GST-LMO2融合蛋白分别用于钓取K562细胞中LMO2的结合蛋白, 结果显示二者都可以结合K562细胞中内源性的GATA1蛋白, 而MBP-LMO2融合蛋白捕获的GATA1蛋白明显多于复性后的GST-LMO2融合蛋白。这一结果提示, 在研究一些分子量小、疏水性强的蛋白质时改变标签蛋白可能是一种有益的尝试。     

不同蛋白标签对LMO2融合蛋白沉淀实验的影响

融合蛋白沉淀技术是一种用来研究蛋白质相互作用的新的体外实验技术, 通常利用蛋白亲和标签与探针蛋白融合表达来钓取未知相互作用蛋白或验证已知蛋白间的相互作用, 其中以谷胱甘肽巯基转移酶(GST)标签最为常用。LMO2(由LIM only缩写得名, 也称Ttg-2或Rbtn2)是一种小分子量难溶蛋白。利用原核系统分别表达了含有GST和麦芽糖结合蛋白(MBP)两种标签的LMO2融合蛋白, 发现GST-LMO2融合蛋白以包涵体的形式表达, 而MBP-LMO2融合蛋白则能够以可溶形式表达, 而且MBP-LMO2的表达量明显高于GST-LMO2融合蛋白。将可溶性的MBP-LMO2融合蛋白和复性后的GST-LMO2融合蛋白分别用于钓取K562细胞中LMO2的结合蛋白, 结果显示二者都可以结合K562细胞中内源性的GATA1蛋白, 而MBP-LMO2融合蛋白捕获的GATA1蛋白明显多于复性后的GST-LMO2融合蛋白。这一结果提示, 在研究一些分子量小、疏水性强的蛋白质时改变标签蛋白可能是一种有益的尝试。     

Soluble expression of a strong thrombolytic pro-urokinase mutant in Escherichia coli

A recombinant pro-urokinase mutant GZ5-sPA was successfully constructed by fusion of a high fibrin-affinity fragment GZ5 to the N-terminus of the serine protease domain of pro-urokinase (sPA). The fragment GZ5 contains a tetrapeptide GPRP and a tripeptide RGD, and was synthesized in bacterial preferred expressing codons. The mutant was then fused to the C-terminus of maltose binding protein (MBP) carried by pMAL-C2x vector, and expressed in Escherichia coli strain Origami (DE3). The produced fusion protein was highly soluble in the cytoplasm of the bacteria. After being cleaved with PreScission Protease to remove MBP tag, GZ5-sPA showed a molecular weight of 31 kDa on SDS-PAGE. GZ5-sPA maintained the same epitope as wild-type pro-urokinase and possessed a thrombolytic activity three times higher than standard urokinase did after being activated as two-chain form. The results could be a clue to other complicated heterogenous proteins similar to pro-urokinase.     

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.     

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签（EDA标签、MBP标签和GST标签）以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。     

重组SARS冠状病毒M蛋白的表达、纯化及鉴定

SARS冠状病毒是人的严重急性呼吸综合征的病原体。根据对其他种类冠状病毒的研究结果 ,膜蛋白 (M蛋白 )是病毒主要的结构蛋白 ,重组M蛋白可被用来作为抗原检测对应冠状病毒的感染和制备疫苗。SARS病毒M蛋白基因克隆到原核表达载体pMAL cRI中 ,利用N端和C端分别融合麦芽糖结合蛋白 (maltosebindingprotein和MxeGyrAinteinCBD的策略 ,在大肠杆菌中初步表达了重组M蛋白 ,并通过Western印迹和质谱对蛋白质进行了鉴定。重组蛋白质经亲和层析得到了部分纯化 ,纯化后的蛋白质将用于功能研究与诊断试剂盒的研制。     

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.