Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Patch cloning method for multiple site-directed and saturation mutagenesis.

BACKGROUND: Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis. RESULTS: To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact. CONCLUSIONS: MUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.

The regulatory C-terminal domain of subunit ε of F0F1 ATP synthase is dispensable for growth and survival of Escherichia coli.

The C-terminal domain of subunit ε of the bacterial F0F1 ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.

Molecular events involved in a single cycle of ligand transfer from an ATP binding cassette transporter, LolCDE, to a molecular chaperone, LolA.

An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters mediating the transmembrane flux of substrates, the LolCDE complex catalyzes the extrusion of lipoproteins anchored to the outer leaflet of the inner membrane. Moreover, the LolCDE complex is unique in that it can be purified as a liganded form, which is an intermediate of the lipoprotein release reaction. Taking advantage of these unique properties, we established an assay system that enabled the analysis of a single cycle of lipoprotein transfer reaction from liganded LolCDE to LolA in a detergent solution. The LolA-lipoprotein complex thus formed was physiologically functional and delivered lipoproteins to the outer membrane in a LolB-dependent manner. Vanadate, a potent inhibitor of the lipoprotein release from proteoliposomes, was found to inhibit the release of ADP from LolCDE. However, a single cycle of lipoprotein transfer occurred from vanadate-treated LolCDE to LolA, indicating that vanadate traps LolCDE at the energized state.

Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunits.

The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 degrees C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 degrees C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits.

A novel ligand bound ABC transporter, LolCDE, provides insights into the molecular mechanisms underlying membrane detachment of bacterial lipoproteins.

The LolCDE complex of Escherichia coli belongs to the ABC transporter superfamily and initiates the lipoprotein sorting to the outer membrane by catalysing their release from the inner membrane. LolC and/or LolE, membrane subunits, recognize lipoproteins anchored to the outer surface of the inner membrane, while LolD hydrolyses ATP on its inner surface. We report here that ligand-bound LolCDE can be purified from the inner membrane in the absence of ATP. Liganded LolCDE represents an intermediate of the release reaction and exhibits higher affinity for ATP than the unliganded form. ATP binding to LolD weakens the interaction between LolCDE and lipoproteins and causes their dissociation in a detergent solution, while lipoprotein release from membranes requires ATP hydrolysis. Liganded LolCDE thus reveals molecular events brought about through ATP binding and hydrolysis. LolCDE is the first example of an ABC transporter purified with tightly bound native substrates. A single molecule of lipoprotein is found to bind per molecule of the LolCDE complex.

Mechanisms underlying energy-independent transfer of lipoproteins from LolA to LolB, which have similar unclosed {beta}-barrel structures.

The Lol system, comprising five Lol proteins, transfers lipoproteins from the inner to the outer membrane of Escherichia coli. Periplasmic LolA accepts lipoproteins from LolCDE in the inner membrane and immediately transfers them to LolB, a receptor anchored to the outer membrane. The unclosed beta-barrel structures of LolA and LolB are very similar to each other and form hydrophobic cavities for lipoproteins. The lipoprotein transfer between these similar structures is unidirectional and very efficient, but requires no energy input. To reveal the mechanisms underlying this lipoprotein transfer, Arg and Phe at positions 43 and 47, respectively, of LolA were systematically mutagenized. The two residues were previously found to affect abilities to accept and transfer lipoproteins. Substitution of Phe-47 with polar residues inhibited the ability to accept lipoproteins from the inner membrane. No derivatives caused periplasmic accumulation of lipoproteins. In contrast, many Arg-43 derivatives caused unusual periplasmic accumulation of lipoproteins to various extents. However, all derivatives, except one having Leu instead of Arg, supported the growth of cells. All Arg-43 derivatives retained the ability to accept lipoproteins from the inner membrane, whereas their abilities to transfer associated lipoproteins to LolB were variously reduced. Assessment of the intensity of the hydrophobic interaction between lipoproteins and Arg-43 derivatives revealed that the LolA-lipoprotein interaction should be weak, otherwise lipoprotein transfer to LolB is inhibited, causing accumulation of lipoproteins in the periplasm.

Reduction of antigenicity of Cry j I, major allergen of Japanese cedar pollen, by the attachment of polysaccharides.

An attempt was made to mask the allergenic structure of a major allergen protein, Cry j I (CJI), in Japanese cedar pollen using the Maillard-type polysaccharide conjugation. The SDS-PAGE pattern of the CJI-galactomannan conjugate prepared by the Maillard reaction showed broad bands widely distributed from 50 kDa to more than 100 kDa, suggesting the attachment of galactomannan. The competitive enzyme-linked immunosorbent assay showed that the IgE antibody in the sera of cedar pollen-sensitive patients reacted strongly with CJI, while it did not react with the CJI-galactomannan conjugate. This result suggests that the antigenicity of CJI is greatly reduced by the conjugation with galactomannan.