Yasufumi Shima

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.

Evolutionary molecular engineering by random elongation mutagenesis

We describe a new method of random mutagenesis that employs the addition of peptide tails with random sequences to the C–terminal of enzyme molecules. A mutant population of catalase I from Bacillus stearothermophilus prepared by this method has a diversity in thermostability and enzyme activity equal to that obtained after random point mutagenesis. When a triple mutant of catalase I (I108T/D130N/I222T)—the thermostability of which is much lower than that of the wild type—was subjected to random elongation mutagenesis, we generated a mutant population containing only mutants with higher thermostability than the triple mutant. Some had an even higher stability than the wild–type enzyme, whose thermostability is considered to be optimized. These results indicate that peptide addition expands the protein sequence space resulting in a new fitness landscape. The enzyme can then move along the routes of the new landscape until it reaches a new optimum. The combination of random elongation mutagenesis with random point mutagenesis should be a useful approach to the in vitro evolution of proteins with new properties.

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.

Characterization of soluble artificial proteins with random sequences

The structural and catalytic properties of two soluble random proteins, RP3‐42 and RP3‐45, of 141 amino acid residues were investigated. Although no marked secondary structure was detected by CD spectrum, sedimentation equilibrium and small‐angle X‐ray scattering studies showed that they form an oligomeric structure and are as compact as the molten globule. The random proteins have low but distinct esterase activity; the values of the second‐order rate constant for the hydrolysis of p‐nitrophenol were 0.78 and 1.39 M−1 s−1 for RP3‐42 and RP3‐45, respectively. The differences in the properties of the random and the native proteins are discussed from the evolutionary point of view.

Sequence and properties of β‐xylosidase from Bacillus pumilus IPO

The nucleotide sequence of the beta-xylosidase (xynB) gene from Bacillus pumilus has been reported previously [Moriyama, H., Fukusaki, E., Crespo, J.C., Shinmyo, A. & Okada, H. (1987) Eur. J. Biochem. 166, 539-545]. However, the sequence identified in the present study is quite different from the previously reported one. The total length of the PstI--EcoRI fragment of a plasmid pOXN295 containing the xynB gene is 2201 bp from our sequencing, while the length of the fragment in the previous data was 2466 bp. The sequences are similar in the N-terminal (500 bp) and C-terminal (260 bp) regions, but those in the central region are completely different. From the following observations, the previous sequence seems to have no reliable experimental basis. First, the restriction sites observed for pOXN295 are quite different from the sites deduced from the sequence. Second, the amino acid composition deduced from the sequence and the composition identified by amino acid analysis of the purified beta-xylosidase are very different. It is confirmed, on the other hand, that our new sequence agrees well with these experimental data. The enzyme was purified to homogeneity from Bacillus pumilus and Escherichia coli harboring a hybrid plasmid which highly expresses the xynB gene. The molecular mass of the enzyme was estimated to be 190 kDa by high performance gel filtration chromatography using TSK-G3000SW and 56 kDa by SDS/polyacrylamide gel electrophoresis. The pH optimum was 7.0, and the optimum temperature was 40 degrees C. The Vm value was estimated to be 1.23 +/- 0.14 mukat/mg (or p-nitrophenyl beta-D-xyloside) and 0.14 +/- 0.011 mukat/mg (for xylobiose), while Km was estimated to be 3.9 +/- 0.59 mM (for p-nitrophenyl beta-D-xyloside) and 8.9 +/- 1.19 mM (for xylobiose).

Importance of compartment formation for a self-encoding system

A self-encoding system designed to have strict “compartition” of the molecules, i.e., to contain only a single molecule of DNA in each compartment, was established, and its evolutionary fate was analyzed. The system comprised the Thermus thermophilus DNA polymerase gene as the informational molecule and its protein product replicating the gene as the functional molecule. Imposing strict compartition allows the self-encoding system to last up to at least the tenth generation, whereas the system ceased to work after the third generation when loose compartition initiated with 100 molecules was imposed. These results provide experimental evidence on the importance of compartition for the maintenance of a self-encoding system. In addition, the extent of diversity in self-replication activity of the compartments was found to be another vital difference in the evolutionary dynamics between the strict and loose compartitions. Although the system with strict compartition provides widely diversified activity of the compartments at each generation, the values of the activity diverge only within a small range in the system with loose compartition. When the variety in the activity of a compartment is small, functional selection becomes weak, and to conform Darwinian evolution may become unfeasible. Therefore, strict compartition is essential for the evolvability of a self-encoding system.

Construction and characterization of phage libraries displaying artificial proteins with random sequences

Three phage libraries, PL1, PL2, and PL3, displaying artificial proteins with random sequences were constructed. The artificial proteins, which are model of ancestral proteins, are derivatives of the 25 kinds of random proteins with about 140 amino acid residues produced via random mutagenesis and combinatorial recombination. The random proteins were displayed on the surface of filamentous bacteriophage as fusion protein with the pIII coat protein at an estimated average number on the phage particles in PL1, PL2, and PL3 of 0.32, 0.32, and 0.08, respectively. Each library was shown to express 10(5) to 10(6) kinds of random proteins. With the phage libraries displaying long random peptides, we now have an effective selection system to observe in vitro evolution of new functional proteins from artificial proteins with random sequences.

Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract Various plasmids harboring the truncated DNA polymerase gene ( polA ) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable Tth Δ NF 2 polymerase [the gene product of polA Δ NF 2, which lacked a 751-bp region (region flanked by initiation codon and Fsp I site in the polA gene)] was selected, and purified from the recombinant Escherichia coli . SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the Tth Δ NF 2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the Tth Δ NF 2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth Δ NF 2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth Δ NF 2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.

Purification and characterization of Qβ replicase with a His-tag

A His-tag was added to the C-terminal of Qβ replicase, an RNA-dependent RNA polymerase of RNA coliphage Qβ, to facilitate enzyme purification. The purified His-tagged enzyme assumed almost the same template specificity as the wild type purified by a conventional method when MDV-poly(+) RNA or Qβ RNA was used as the template. Here, we showed the efficiency of the approach surmounts the present available ones. The availability of Qβ replicase of quality affords its implementation for the synthesis and amplification of RNA molecules as well as further elucidation on the molecular mechanism of the enzyme reaction.