Itaru Urabe

Synthesis of functional protein in liposome.

The liposome consisting of eggPC, cholesterol, and DSPE-PEG5000 with a molar ratio of 1.5:1:0.08 was used to entrap cell-free protein synthesis reaction mixture. The synthesis of a mutant green fluorescent protein in the liposome was confirmed by the fluorescence emitted from the liposome on flow cytometry analysis and fluorescence microscopy. The protein synthesized in the liposome is hence functional.

Expression of a cascading genetic network within liposomes.

Liposomes have long been used as possible compartments for artificial cells, and it has been shown that liposomes can sustain various types of biochemical reactions. To elevate the degree of molecular complexity of the system in liposomes, we have constructed a two‐stage genetic network encapsulated in liposomes. This two‐stage genetic network was constructed with the plasmid pTH, in which the protein product of the first stage (T7 RNA polymerase) is required to drive the protein synthesis of the second stage (GFP). We show that the two‐stage genetic network constructed in a cell‐free expression system is functional within liposomes.

Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence‐labeled PEG as a test macromolecule. The size of Fluorescence‐labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence‐labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.

Replication of Genetic Information with Self-Encoded Replicase in Liposomes

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a “self‐encoding system”, in which the genetic information is replicated by self‐encoded replicase in liposomes. That is, the universal reaction was reconstituted within a microcompartment bound by a lipid bilayer. The system was assembled by using one template RNA sequence as the information molecule and an in vitro translation system reconstituted from purified translation factors as the machinery for decoding the information. In this system, the catalytic subunit of Qβ replicase is synthesized from the template RNA that encodes the protein. The replicase then replicates the template RNA that was used for its production. This in‐liposome self‐encoding system is one of the simplest such systems available; it consists of only 144 gene products, while the information and the function for its replication are encoded on different molecules and are compartmentalized into the microenvironment for evolvability.

Stabilization of xylanase by random mutagenesis.

Four heat‐resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser‐26 to Trp, Gly‐38 to Asp and Thr‐126 to Ser; N102, Gly‐38 to Asp; N104, Gly‐38 to Ser and Arg‐48 to Lys; F1, Ser‐12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

Reaction of glutaraldehyde with amino and thiol compounds

Abstract Stoichiometry, pH dependence, and reversibility of the reaction of glutaraldehyde with various amino and thiol compounds were investigated to elucidate the chemical nature of glutaraldehyde. For glutaraldehyde, three commercial samples were examined. They have different spectral characteristics probably due to the difference in the content of α, β-unsaturated aldehyde polymers formed by aldol condensations of glutaraldehyde, but the amount of such unsaturated structures is very small, and the chemical reactivity of these samples are almost the same. Therefore, the chemical reactivity characteristic of glutaraldehyde is not due to the α, β-unsaturated aldehydes. Glutaraldehyde reacts with the amino group in a wide pH range ( ≧ pH 3 ). The reactions at pH 7 and 9 are almost irreversible, though a little reversibility is observed. The reaction rate becomes very slow after the initial rapid phase. The average molar ratio of the amino and aldehyde groups consumed during the reaction of glutaraldehyde with the amino group is in a range of 0.3-0.2. Glutaraldehyde reacts with cysteine with a stoichiometric relationship of one mol of the thiol and amino groups of cysteine per mol of glutaraldehyde. Glutaraldehyde does not react with the thiol group without the presence of the primary amino group; the average stoichiometric relationship is 0.5–0.6 mol of the thiol group, about 0.4 mol of the amino group, and 1 mol of glutaraldehyde, under the conditions of excess in the thiol and amino groups. These results indicate the complex nature of the glutaraldehyde reaction.

Experimental rugged fitness landscape in protein sequence space.

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12–130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7×104-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffmans n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18–24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.

Solubility of artificial proteins with random sequences

A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The soluble random proteins RP3–42 and RP3–45 and insoluble RP3–70 were purified. The solubility of the purified form is the same as that in the cell lysate.

Preparation of polyethylene glycol‐bound NAD and its application in a model enzyme reactor

Recently many kinds of water-soluble macromolecular NAD derivatives have been prepared and their coenzymatic properties and applications in biochemical reactors have been reported [l-4]. However, there have been no reports of systematic investigations about the structure and cofactor activity of macromolecular NAD derivatives. We have synthesized a NAD derivative carrying a vinyl group [5]. The NAD derivative was copolymerized with acrylamide or methacrylamide and various kinds of water-soluble macromolecular NAD derivatives (polymeric NAD derivatives) were obtained [5-71. Based on the results of the investigation into the coenzymatic properties of the polymeric NAD derivatives, it was suggested that an NAD derivative with a smaller molecular size and a lower NAD content in the polymer chain would have higher cofactor activity. Indeed, a polymeric NAD derivative prepared by copolymeri~ation with me~ac~l~ide had good cofactor activity [6], but the polymeric NAD derivative was not suitable for application in enzyme reactors because of its low NAD content in the total polymer and the wide distribution of its molecular size. Therefore, we planned to make a new watersoluble macromolecular NAD derivative with a fvted NAD content and an appropriate molecular size. From among soluble polymers suitable for binding of NAD, we used polyethylene glycol, which was commercially available in a variety of molecular weights (1500-20 000) and had only two functional groups at its ends. Here, polyethylene glycol-bound NAD (PEG-NAD) was prepared by coupling of Nd-(2-car-

Evolution of an arbitrary sequence in solubility

We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His6 tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation. Analysis of the synonymous and nonsynonymous mutations found in the deduced amino acid sequence of the selected polypeptides revealed that selection had accelerated the evolutionary rate. The solubility and hydrophobicity of the polypeptides and the 25 arbitrarily chosen random polypeptides found in a previously prepared library were determined, analyzed, and interpreted from the landscape on the protein sequence space. This study showed the evolvability of an insoluble arbitrary sequence toward a soluble one, hence, it provides a new perspective on the field of artificial evolution.