Towards the Creation of Novel Proteins by Block Shuffling

Abstract

We have been investigating the creation of novel proteins by means of block shuffling, where the term block refers to an amino acid sequence that corresponds to particular features of proteins, such as secondary structures, modules, functional motifs, and so on. Block shuffling makes it possible to explore the global sequence space, which is not feasible with conventional methods, such as DNA shuffling or family shuffling. To investigate what properties are required for the building blocks, we have analyzed the foldability and enzymatic activity of barnase mutants obtained by permutation of modules or secondary structure units. This reconstructive approach indicated that secondary structure units with mutual long-range interactions are more suitable than modules as building blocks, at least in the case of barnase. The results also suggested that proteins in evolutionarily intermediate states are created by block shuffling, and such proteins have the potential to be evolved into mature globular proteins. For the construction of combinatorial protein libraries, we have developed random multi-recombinant PCR (RM-PCR), which can combine different DNA fragments without homologous sequences. The libraries can be utilized for in vitro selection using in vitro virus (mRNA display) or stable (DNA display), which have also been developed in our laboratory. In this review article, we summarize our strategy to create novel proteins by block shuffling and review key literature in the field. Possible applications of the block shuffling strategy are also discussed.

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“…Following a related idea, it has been suggested that new enzymes can be generated by recombining different segments of modern proteins, and the concept has been shown to be successful. 4,23 Here we describe Systematic Catalytic Loop Exchange (SCLE), a new strategy for exchanging loops on a TIM barrel fold. As a scaffold, we chose a variant of phosphoribosylanthranilate isomerase (PRAI-LoxP) from Escherichia coli, a monomeric enzyme where we have previously done significant work.…”

Exploration of the Origin and Evolution of Globular Proteins by mRNA Display

“…When blocks including secondary structures such as α-helix or β-sheet were combined as "structural motifs", greater structure forming capability and enzymatic activity were found. 52,75,76 Recently, several methods have been developed to combine multiple DNA fragments without homologous sequences. Structure-based combinatorial protein engineering (SCOPE) employs chimeric primers to combine structural elements from different proteins that have similar folds but have a low level of sequence identity.…”

“…In particular, I next focus attention on the application of mRNA display to the selection of functional proteins from fully randomized and partially randomized libraries with a limited set of amino acids, , and selection of functional proteins from block-shuffled libraries, − which are constructed on the basis of the hypotheses described above. Such application of mRNA display allows the validity of the hypotheses to be evaluated in terms of the numbers of functional proteins obtained from these libraries.…”

On the Adaptive Design Rules of Biochemical Networks in Evolution

“…The proposed robust adaptive design rules of natural selection also provide a systematic robust biochemical circuit design method of biochemical networks for drug design and robust engineered synthetic biocircuit design purposes in the future. We can use computational prediction and rational design ( Altamirano et al 2000 ; Johannes et al 2005 ; Tsuji et al 2006 ), directed evolution ( May et al 2000 ; Wang et al 2000 ; Yuan et al 2005 ) and dynamic controller ( Farmer and Liao, 2000 ; Bulter et al 2004 ) to quickly create a library of variants for artificial evolution to achieve the desired property of biochemical networks. Rational design and directed evolution are to modify the catalytic or binding property of an enzyme which corresponds to the changes of kinetic parameters g ij and h ij in the S-System model through modulating the enzyme structure and through DNA shuffling, respectively.…”