Maltose-binding protein (MBP) is a part of the

maltose } Maltose ( or ), also known as maltobiose or malt sugar, is a disaccharide formed from two units of glucose joined with an α(1→4) bond. In the isomer isomaltose, the two glucose molecules are joined with an α(1→6) bond. Maltose is the tw ...

maltodextrin Maltodextrin is a name shared by two different families of chemicals. Both families are glucose polymers (also called ''dextrose polymers'' or ''Dextrin, dextrins''), but have little chemical or nutritional similarity. The digestible maltodextr ...

system of ''

Escherichia coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...

'', which is responsible for the uptake and efficient

catabolism Catabolism () is the set of metabolic pathways that breaks down molecules into smaller units that are either oxidized to release energy or used in other anabolic reactions. Catabolism breaks down large molecules (such as polysaccharides, lipid ...

of maltodextrins. It is a complex regulatory and transport system involving many

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s and protein complexes. MBP has an approximate molecular mass of 42.5 kilodaltons.

Structure and folding

MBP is encoded by the ''malE''

gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...

of ''Escherichia coli''. The ''malE'' gene codes for a precursor polypeptide (396

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

residues) which yields the mature MBP (370 residues) upon cleavage of the NH₂-terminal extension (26 residues). The precursor and mature forms of MBP do not contain any

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

residues. MBP is a monomeric protein. Crystal structures have shown that MBP is divided into two distinct globular domains that are connected by three short polypeptide segments. The two domains are separated by a deep groove that contains the maltose/maltodextrin binding site. Comparison of the structures of the liganded and unliganded forms of MBP has shown that the binding of maltose induces a major

conformational change In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors. A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or othe ...

that closes the groove by a rigid motion of the two domains around the linking polypeptide hinge. Both precursor and mature forms of MBP are functional for the binding of maltose. The NH₂-terminal extension decreases the

folding Fold, folding or foldable may refer to: Arts, entertainment, and media * ''Fold'' (album), the debut release by Australian rock band Epicure * Fold (poker), in the game of poker, to discard one's hand and forfeit interest in the current pot *Abov ...

rate of the precursor form of MBP relative to its mature form by at least 5 fold, but it has no effect on the unfolding rate. The equilibrium unfolding of MBP can be modelled by a two-state mechanism with a stability ∆G(H₂O) equal to 9.45 kcal mol⁻¹ at 25 °C, pH 7.6.

Localization and export

MBP is exported into the

periplasmic space The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in Gram-negative (more accurately "diderm") bacteria. Using cryo-electron microsco ...

of ''E. coli''. The NH₂-terminal extension of MBP, also termed

signal peptide A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short peptide (usually 16–30 amino acids long) present at the ...

, has two roles: (i) it slows down folding of the newly synthesized polypeptide, and (ii) it directs this polypeptide to the membrane and SecYEG

translocon The translocon (also known as a translocator or translocation channel) is a complex of proteins associated with the translocation of polypeptides across membranes. In eukaryotes the term translocon most commonly refers to the complex that transpor ...

. Once folded, the precursor can no longer enter the translocation pathway. The introduction of a charged amino-acid residue or a

proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...

residue within the hydrophobic core of the signal peptide is sufficient to block export. The defective exports of the mutant MBPs are consistent with the alpha-helical conformation and hydrophobic interactions of the signal peptide in its interaction with the translocon motor protein

SecA SECA, SecA or Seca may refer to: * Société Européenne de Contrôle d'Accès, now Nagra France * The SECA Mediaguard encryption system, designed by the above company. * Society for the Encouragement of Contemporary Art, an auxiliary part of the S ...

Control of expression

The ''malE'' gene, coding for MBP, belongs to the

Mal regulon In bacterial genetics, the mal regulon is a regulon - or group of genes under common regulation - associated with the catabolism of maltose and maltodextrins. The system is especially well characterized in the model organism ''Escherichia coli'', wh ...

of ''E. coli'', which consists of ten genes whose products are geared for the efficient uptake and utilization of

and maltodextrins. All the gene involved in the transport of maltose/maltodextrin, including ''malE'', are clustered in the ''malB'' region of ''E. coli'' and organized in two divergent

operons In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...

: ''malE-malF-malG'' and ''malK-lamB''. The

transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, often th ...

start sites at the ''malEp'' and ''malKp'' promoters are distant of 271 base pairs. The ''malEp'' and ''malKp'' promoters are synergistically activated by protein MalT, the activator of the Mal regulon and by the

cAMP receptor protein cAMP receptor protein (CRP; also known as catabolite activator protein, CAP) is a regulatory protein in bacteria. Protein CRP protein binds cyclic adenosine monophosphate (cAMP), which causes a conformational change that allows CRP to bind tigh ...

CRP. This activation is a coupled process that involves, going from ''malEp'' towards ''malKp'': two MalT binding sites; three CRP binding sites, and two overlapping sets of three MalT binding sites, staggered by three base pairs. Transcription activation requires the binding of

adenosine triphosphate Adenosine triphosphate (ATP) is a nucleoside triphosphate that provides energy to drive and support many processes in living cell (biology), cells, such as muscle contraction, nerve impulse propagation, and chemical synthesis. Found in all known ...

(ATP) and

maltotriose Maltotriose is a trisaccharide (three-part sugar) consisting of three glucose molecules linked with α-1,4 glycosidic bonds. It is most commonly produced by the digestive enzyme alpha-amylase (a common enzyme in human saliva) on amylose in star ...

to MalT and the binding of

cyclic AMP Cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3',5'-cyclic adenosine monophosphate) is a second messenger, or cellular signal occurring within cells, that is important in many biological processes. cAMP is a derivative of adenosine triph ...

to the dimer of CRP. The unliganded form of MalT is monomeric whereas its liganded form, in the presence of ATP and maltotriose, is oligomeric.

Use as a protein and peptide vector

MBP is used to increase the solubility of

recombinant protein Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the ...

s expressed in ''E. coli''. In these systems, the protein of interest is often expressed as a MBP-

fusion protein Fusion proteins or chimeric (kī-ˈmir-ik) proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this '' fusion gene'' ...

, preventing aggregation of the protein of interest. The mechanism by which MBP increases solubility is not well understood. In addition, MBP can itself be used as an

affinity tag Affinity may refer to: Commerce, finance and law * Affinity (law), kinship by marriage * Affinity analysis, a market research and business management technique * Affinity Credit Union, a Saskatchewan-based credit union * Affinity Equity Part ...

for

purification Purification is the process of rendering something pure, i.e. clean of foreign elements and/or pollution, and may refer to: Religion * Ritual purification, the religious activity to remove uncleanliness * Purification after death * Purification ...

of recombinant proteins. The fusion protein binds to

amylose Amylose is a polysaccharide made of α-D-glucose units, bonded to each other through α(1→4) glycosidic bonds. It is one of the two components of starch, making up approximately 20–25% of it. Because of its tightly packed Helix, helical struct ...

columns A column or pillar in architecture and structural engineering is a structural element that transmits, through compression, the weight of the structure above to other structural elements below. In other words, a column is a compression member ...

while all other proteins flow through. The MBP-protein fusion can be purified by eluting the column with maltose. Once the fusion protein is obtained in purified form, the protein of interest is often cleaved from MBP with a specific

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalysis, catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products ...

and can then be separated from MBP by

affinity chromatography Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...

. A first study of the relations between structure and functions of MBP was performed by random insertion of a short DNA fragment, coding for a

BamHI BamHI (pronounced "Bam H one") (from ''Bacillus amyloliquefaciens'') is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site. This exhibit focuses o ...

restriction site In molecular biology, restriction sites, or restriction recognition sites, are regions of a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides; these are recognized by restriction enzymes, which cleave the DNA at ...

, into the ''malE'' gene. Some of the insertions affected the functions of MBP whereas others were permissive. The permissive sites that were internal to MBP, were used to insert antigenic peptides and challenge the immune response in mice. The 3'-OH terminal insertions were used to create fusion proteins and develop the use of MBP as an affinity handle for the purification of foreign proteins and peptides by affinity chromatography on cross-linked amylose and elution with maltose in mild physico-chemical conditions. Several

plasmid vector A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...

s were developed to facilitate the expression and purification of such fusion proteins. When the recombinant MBP includes a

, the fusion protein can be exported into the

, which facilitates its purification since the periplasmic fluid contains only a limited number of proteins and can be recovered either by an

osmotic shock Osmotic shock or osmotic stress is physiologic dysfunction caused by a sudden change in the solute concentration around a cell, which causes a rapid change in the movement of water across its cell membrane. Under hypertonic conditions - conditio ...

or by permeabilization of the

bacterial outer membrane The bacterial outer membrane is found in gram-negative bacteria. Gram-negative bacteria form two lipid bilayers in their cell envelopes - an inner membrane (IM) that encapsulates the cytoplasm, and an outer membrane (OM) that encapsulates the p ...

with

antibiotics An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...

such as

Polymyxin B Polymyxin B, sold under the brand name Poly-Rx among others, is an antibiotic used to treat meningitis, pneumonia, sepsis, and urinary tract infections. While it is useful for many Gram negative infections, it is not useful for Gram positive infe ...

. Such an export of the fusion protein into the periplasmic space enables the formation of

disulfide bond In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In inor ...

s in the passenger protein, for example

antibody An antibody (Ab) or immunoglobulin (Ig) is a large, Y-shaped protein belonging to the immunoglobulin superfamily which is used by the immune system to identify and neutralize antigens such as pathogenic bacteria, bacteria and viruses, includin ...

fragments. Foreign proteins that are exported or secreted in their native organism, can usually be exported into the ''E. coli'' periplasm by fusion with MBP. Examples of cytoplasmic proteins that could be exported by fusion with MBP, include the monomeric

Klenow polymerase The Klenow fragment is a large protein fragment produced when DNA polymerase I from ''E. coli'' is enzyme, enzymatically cleaved by the protease subtilisin. First reported in 1970, it retains the 5' → 3' polymerase activity and the 3’ → 5� ...

and the dimeric Gene V protein of phage M13. When the recombinant MBP includes either a defective or no signal peptide the fusion protein remains within the bacterial

cytoplasm The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell a ...

from where it can be recovered by breaking open the cells. The fusion of proteins with MBP usually enhances their solubility and facilitates their proper folding so that the fusion proteins are most often bifunctional. In addition, such fusions can facilitate the crystallisation of difficult proteins, e.g. membrane proteins. The crystallized protein can often have their structures solved by

X-ray crystallography X-ray crystallography is the experimental science of determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to Diffraction, diffract in specific directions. By measuring th ...

using

molecular replacement Molecular replacement (MR) is a method of solving the phase problem in X-ray crystallography. MR relies upon the existence of a previously solved protein structure which is similar to our unknown structure from which the diffraction data is deriv ...

on a known MBP structure.

References

External links

N-Terminal Fusion of Target Protein to Maltose-Binding Protein
at

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