Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries

BACKGROUND: We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis. RESULTS: A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 10(16 )diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons. CONCLUSIONS: Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases

In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro

AbstractAdequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of ‘in vitro virus’ was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was ∼10%, thus indicating a population of the in vitro virus to have ∼1012 protein variants, this number being 104 that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions

Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer

<p>Abstract</p> <p>Background</p> <p>The microarray has contributed to developing the omic analysis. However, as it depends basically on the surface reaction, it is hard to perform bulk reactions and sequential multistep reactions. On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.</p> <p>Results</p> <p>Here, we report a novel microarray technology which enables us to explore advanced applications, termed <it>microarray-with-manageable volumes </it>(MMV). The technical essence is in the pipette-free direct parallel transfer from well to well performed by centrifugation, evading the evaporation and adsorption-losses during handling. By developing the MMV plate, accompanying devices and techniques, generation of multiple conditions (256 kinds) and performance of parallel multistep reactions, including PCR and <it>in vitro tr</it>anslation reactions, have been made possible. These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.</p> <p>Conclusions</p> <p>With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible.</p

Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA

A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA–protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA–protein fusion is ligated to the 3′ ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA–protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of ‘RNA chip-to-protein chip’ using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution

An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion (=viral particle)” (mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated

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 1.7×10(4)-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 Kauffman's 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