Some of the insertions affected the functions of MBP whereas others were permissive. Once the fusion protein is obtained in purified form, the protein of interest is often cleaved from MBP with a specific protease and can then be separated from MBP by affinity chromatography.Ī 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 restriction site, into the malE gene. The MBP-protein fusion can be purified by eluting the column with maltose. The fusion protein binds to amylose columns while all other proteins flow through. In addition, MBP can itself be used as an affinity tag for purification of recombinant proteins. The mechanism by which MBP increases solubility is not well understood. In these systems, the protein of interest is often expressed as a MBP- fusion protein, preventing aggregation of the protein of interest. MBP is used to increase the solubility of recombinant proteins expressed in E. The unliganded form of MalT is monomeric whereas its liganded form, in the presence of ATP and maltotriose, is oligomeric. Transcription activation requires the binding of adenosine triphosphate (ATP) and maltotriose to MalT and the binding of cyclic AMP to the dimer of CRP. This activation is a coupled process that involves, going from malEp towards malKp: two MalT binding sites three CRP binding site, and two overlapping sets of three MalT binding sites, staggered by three 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 CRP. The transcription start sites at the malEp and malKp promoters are distant of 271 base pairs.
coli and organized in two divergent operons: malE-malF-malG and malK-lamB. All the gene involved in the transport of maltose/maltodextrin, including malE, are clustered in the malB region of E. coli, which consists of ten genes whose products are geared for the efficient uptake and utilization of maltose and maltodextrins. The malE gene, coding for MBP, belongs to the Mal regulon of E. 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. The introduction of a charged amino-acid residue or a proline residue within the hydrophobic core of the signal peptide is sufficient to block export. Once folded, the precursor can no longer enter the translocation pathway. The NH 2-terminal extension of MBP, also termed signal peptide, 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. MBP is exported into the periplasmic space of E. The equilibrium unfolding of MBP can be modelled by a two-state mechanism with a stability ∆G(H 2O) equal to 9.45 kcal mol −1 at 25 ☌, pH 7.6. The NH 2-terminal extension decreases the folding 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. īoth precursor and mature forms of MBP are functional for the binding of maltose. Comparison of the structures of the liganded and unliganded forms of MBP has shown that the binding of maltose induces a major conformational change that closes the groove by a rigid motion of the two domains around the linking polypeptide hinge. The two domains are separated by a deep groove that contains the maltose/maltodextrin binding site. Crystal structures have shown that MBP is divided into two distinct globular domains that are connected by three short polypeptide segments. The precursor and mature forms of MBP do not contain any cysteine residues. The malE gene codes for a precursor polypeptide (396 amino acid residues) which yields the mature MBP (370 residues) upon cleavage of the NH 2-terminal extension (26 residues). MBP is encoded by the malE gene of Escherichia coli.