Maltose-binding Protein Domain Research Articles

Recombinant Human Erythropoietin with Additional Processable Protein Domains: Purification of Protein Synthesized in Escherichia coli Heterologous Expression System.

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.

Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris

The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins.

Modulation of effector affinity by hinge region mutations also modulates switching activity in an engineered allosteric TEM1 β-lactamase switch

RG13 is an engineered allosteric β-lactamase (BLA) for which maltose is a positive effector. RG13 is a hybrid protein between TEM1 BLA and maltose-binding protein (MBP). Maltose binding to MBP is known to convert the open form of the protein to the closed form through conformational changes about the hinge region. We have constructed and genetically selected several variants of RG13 modified in the hinge region of the MBP domain and explored their effect on β-lactam hydrolysis, maltose affinity and maltose-induced switching. Hinge mutations that increased maltose affinity the most (and thus presumably close the apo-MBP domain the most) also abrogated switching the most. We provide evidence for a model of RG13 switching in which there exists a threshold conformation between the open to closed form of the MBP domain that divides states that catalyze β-lactam hydrolysis with different relative rates of acylation and deacylation.

Enzymatic Redesigning of Biologically Active Heparan Sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

The Protozoan Parasite Cryptosporidium parvum Possesses Two Functionally and Evolutionarily Divergent Replication Protein A Large Subunits

Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.

Separation of MBP fusion proteins through affinity membranes.

Cellulose microporous membranes have been modified in order to obtain a stationary phase specific for the recovery of a class of fusion proteins containing the maltose binding protein domain, through affinity chromatography separations. The feasibility of a single step separation process for the recovery of large amounts of the desired product has been considered. To that purpose, a preparative scale module has been realized, suitable for flat sheet membranes. The affinity matrix used proved to be highly selective toward the fusion proteins examined. The binding capacity determined is comparable with the nominal binding capacity of commercially available supports. The influence of the relevant working parameters, such as flow rate, on the performances of the recovery process has been studied.

Evidence That the Transmembrane Domain Proximal Region of the Human T-cell Leukemia Virus Type 1 Fusion Glycoprotein gp21 Has Distinct Roles in the Prefusion and Fusion-activated States

To investigate the structural context of the fusion peptide region in human T-cell leukemia virus type 1 gp21, maltose-binding protein (MBP) was used as an N-terminal solubilization partner for the entire gp21 ectodomain (residues 313-445) and C-terminally truncated ectodomain fragments. The bacterial expression of the MBP/gp21 chimeras resulted in soluble trimers containing intramonomer disulfide bonds. Detergents blocked the proteolytic cleavage of fusion peptide residues in the MBP/gp21-(313-425) chimera, indicating that the fusion peptide is available for interaction with detergent despite the presence of an N-terminal MBP domain. Limited proteolysis experiments indicated that the transmembrane domain proximal sequence Thr(425)-Ala(439) protects fusion peptide residues from chymotrypsin. MBP/gp21 chimera stability therefore depends on a functional interaction between N-terminal and transmembrane domain proximal regions in a gp21 helical hairpin structure. In addition, thermal aggregation experiments indicated that the Thr(425)-Ser(436) sequence confers stability to the fusion peptide-containing MBP/gp21 chimeras. The functional role of the transmembrane domain proximal sequence was assessed by alanine-scanning mutagenesis of the full-length envelope glycoprotein, with 11 of 12 single alanine substitutions resulting in 1.5- to 4.5-fold enhancements in cell-cell fusion activity. By contrast, single alanine substitutions in MBP/gp21 did not significantly alter chimera stability, indicating that multiple residues within the transmembrane domain proximal region and the fusion peptide and adjacent glycine-rich segment contribute to stability, thereby mitigating the potential effects of the substitutions. The fusion-enhancing effects of the substitutions are therefore likely to be caused by alteration of the prefusion complex. Our observations suggest that the function of the transmembrane domain proximal sequence in the prefusion envelope glycoprotein is distinct from its role in stabilizing the fusion peptide region in the fusion-activated helical hairpin conformation of gp21.

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x 10(4) and 7.1 x 10(4) M(-1) s(-1) were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants (KM) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 microM, respectively. The corresponding value for "wild-type" NR in solution containing TNT was 78 microM, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 microM, and the sensitivities were determined to be 205 and 222 nA/microM, respectively.

Identification of a Topology Control Domain in the Tetracycline Resistance Protein

Two N-terminal fusion proteins combining Escherichia coli maltose-binding protein (MBP) and the 12-transmembrane-segment pBR322 tetracycline resistance protein (Tet) have been constructed to determine the strength and location of topology control signals within the N-terminal portion of the Tet protein. The fusions contain either a secretable (wild type) or a nonsecretable (MBPΔ2-26) MBP domain joined to the normally cytoplasmic N-terminus of the Tet protein. The effects of MBP targeting on Tet topology were investigated by analyzing the susceptibility of fusion strains to tetracycline and by proteolysis of the fusion proteins in inverted membrane vesicles and spheroplasts. The fusion protein containing MBPΔ2-26 conferred tetracycline resistance to the host strain and gave a normal pattern of Tet digestion fragments, indicating that its Tet domain is oriented and folded properly in the membrane. In contrast, the fusion containing secretable MBP was catalytically inactive apparently due to transfer of the Tet N-terminus to the periplasm with MBP. However, protease treatment of this fusion revealed that MBP secretion seems to affect only the topology of segments 1 and/or 2 of the Tet domain. Therefore, a strong topology control sequence appears to be located in the first cytoplasmic loop of the protein.

Expression and Characterization of a Single Recombinant Proteoglycan Tandem Repeat Domain of Link Protein That Binds Zinc and Hyaluronate

The first proteoglycan tandem repeat (PTR) of bovine link protein has been cloned in the pMAL-c vector and overexpressed in fusion with maltose-binding protein (MBP) in Escherichia coli. The fusion protein can be isolated from the soluble phase of the bacterial lysate by amylose affinity chromatography. The PTR domain can be cleaved from the MBP domain with factor Xa protease. Evidence using zinc affinity chromatography is presented which indicates that at least one of the zinc-binding sites of bovine link protein is contained within the first PTR domain. Zinc affinity chromatography was then incorporated as the final purification step of the MBP/PTR protein. Evidence for the binding of MBP/PTR to hyaluronic acid (HA) is demonstrated by coprecipitation with HA using cetylpyridinium chloride. Binding is specific since MBP/PTR does not coprecipitate with chondroitin sulfate. Binding is also demonstrated in an ELISA assay on HA-coated plates. In this assay, binding could be inhibited by the addition of HA or HA oligosaccharides.

In Vitro Interaction of Nitrate-responsive Regulatory Protein NarL with DNA Target Sequences in the fdnG, narG, narK and frdA Operon Control Regions of Escherichia coli K-12

The narL gene product is a nitrate-responsive activator and repressor of anaerobic respiratory gene expression. Mutational studies and sequence comparisons have suggested that NarL protein binding sites contain heptameric sequences related to the consensus, TACYNMT (where Y=C or T, M = A or C, and N = any nucleotide). There are four NarL heptamers in the -105 region of the fdnGHI (formate dehydrogenase-N) operon, and mutational analysis supports the role of these heptamers in nitrate induction.To examine NarL-DNA interactions, we purified the NarL protein as a maltose binding protein (MBP) fusion protein (MBP-NarL). A constitutive mutant form with a single substitution (V88A) in the amino-terminal (response regulator) region was used. The MBP-NarL(V88A) protein protected all four heptamers in the fdnG operon control region from DNase I cleavage. Identical footprints were observed with NarL(V88A) protein that had been proteolytically cleaved free from the MBP domain. Binding of MBP-NarL(V88A) protein to the four heptamers in the - 105 region of the fdnG operon appeared to be cooperative, and occupancy of the central heptamers was necessary for occupancy of the flanking heptamers. In addition to the V88A substitution, a low molecular weight phosphodonor, such as acetyl phosphate, was required for observable footprints. This indicates that phosphorylation of the NarL protein enhances its affinity for its multiple DNA targets in the fdnG operon, perhaps by increasing protein-protein interactions rather than protein-DNA interactions. We also performed footprinting studies at the narGHJI (nitrate reductase), narK (nitrite efflux), and frdABCD (fumarate reductase) operon control regions. Extensive areas of each control region were protected from DNase I attack by phosphorylated MBP-NarL(V88A) protein. The narG operon control region was protected from positions -50 to -110, and, at higher protein concentrations, also around position -200. Mutational analysis indicates that the NarL heptamer centered at position -89, in addition to the previously-identified -200 region, is involved in nitrate induction. Comparisons of the four operon control regions studied indicate that the NarL heptamers are arranged with diverse orientations and spacing.

Membrane Protein Topology Determination by Proteolysis of Maltose Binding Protein Fusions

A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest. Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain. A secreted MBP domain is attached to the protein if its N-terminus resides in the periplasm. A cytosolic MBP domain (MBP Δ2-26) is attached to the protein when its N-terminus resides in the cytoplasm. The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first penplasmic loop. Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP. The mixture of digestion products is analyzed by Western immunoblotting using anti-MHP antiserum to detect cleavage fragments. Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain. The technique has the advantage that neither overexpression of the protein nor high-quality antiserum to it are required for detection of protease fragments. Furthermore, the method can be applied to screen membrane proteins for structure alterations that have been introduced deliberately into them.

CDNA Cloning, Expression in Escherichia coli and Purification of Human 6-Pyruvoyl-Tetrahydropterin Synthase

cDNA clones for human 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the biosynthetic pathway of tetrahydrobiopterin, were isolated from a human Molt-4 cell cDNA library by cross-hybridization with a rat cDNA. One cDNA clone contained the entire coding sequence of 435 base pairs. The cDNA was expressed in Escherichia coli using the expression vector pMAL as a fusion protein with maltose-binding protein. After affinity purification through its maltose-binding protein domain, the fusion protein was digested by factor Xa at a specific cleavage site inserted between the domains. The main product was a protein species with a native molecular mass of 90 kDa and a subunit molecular mass of 17 kDa, and the molecular masses and its kinetic properties were similar to those of the human enzyme purified from the liver.

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor X a protease between the two domains. Cleavage by factor X a separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (β-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.