Nobuhide Doi

STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro

We have developed a new method that permits the complete in vitro construction and selection of peptide or protein libraries. This method relies on an in vitro transcription/translation reaction compartmentalized in water in oil emulsions. In each emulsion compartment, streptavidin (STA)‐fused polypeptides are synthesized and attached to the encoding DNA via its biotin label. The resulting protein‐DNA fusion molecules recovered from the emulsion can be subjected to affinity selection based on the properties of the peptide portion, whose sequence can be determined from that of its DNA‐tag. This method, named ‘STABLE’ (STA‐biotin linkage in emulsions), should be useful for rapid in vitro evolution of proteins and for ligand‐based selection of cDNA libraries.

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution.

Protein‐engineering techniques have been adapted for the molecular design of biosensors that combine a molecular‐recognition site with a signal‐transduction function. The optical signal‐transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand‐binding site. Here we describe a general method of creating entirely new molecular‐recognition sites on GFPs. At the first step, a protein domain containing a desired molecular‐binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 β‐lactamase and its inhibitory protein as our model protein‐ligand system. ‘Allosteric GFP biosensors’ constructed by this method may be used in a wide range of applications including biochemistry and cell biology.

Metabolic Engineering of Carotenoid Biosynthesis in Escherichia coli by Ordered Gene Assembly in Bacillus subtilis

ABSTRACT We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 μg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a pR promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions

We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.

Insertional gene fusion technology.

The classical ‘end to end’ gene fusion technique has widely been used for monitoring gene expression, biological screening and purification of recombinant proteins. Recent progress with the ‘insertional’ gene fusion approach, on the other hand, has demonstrated that this technique can be utilized for membrane protein topology analysis, display of randomized protein libraries and design of biosensor proteins. In this review, we describe examples of insertional gene fusion and compare the old and new gene fusion techniques.

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein–protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein–protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.

Bicistronic DNA display for in vitro selection of Fab fragments

In vitro display methods are superior tools for obtaining monoclonal antibodies. Although totally in vitro display methods, such as ribosome display and mRNA display, have the advantages of larger library sizes and quicker selection procedures compared with phage display, their applications have been limited to single-chain Fvs due to the requirement for linking of the mRNA and the nascent protein on the ribosome. Here we describe a different type of totally in vitro method, DNA display, that is applicable to heterodimeric Fab fragments: in vitro compartmentalization in water-in-oil emulsions allows the linking of an oligomeric protein and its encoding DNA with multiple ORFs. Since previously used emulsions impaired the synthesis of functional Fab fragments, we modified conditions for preparing emulsions, and identified conditions under which it was possible to enrich Fab fragments 106-fold per three rounds of affinity selection. Furthermore, we confirmed that genes encoding stable Fab fragments could be selected from a Fab fragment library with a randomized hydrophobic core in the constant region by applying heat treatment as a selection pressure. Since this method has all advantages of both phage display and totally in vitro display, it represents a new option for many applications using display methods.

Insertion of foreign random sequences of 120 amino acid residues into an active enzyme

Random sequences of 120–130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the proteins function by further mutagenesis simulate one of the events in protein evolution.

Rapid antibody selection by mRNA display on a microfluidic chip

In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 106- to 108-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naïve and randomized single-chain Fv libraries of ∼1012 molecules. Furthermore, we confirmed that not only protein–protein (antigen–antibody) interactions, but also protein–DNA and protein–drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.

SCREENING OF CONFORMATIONALLY CONSTRAINED RANDOM POLYPEPTIDE LIBRARIES DISPLAYED ON A PROTEIN SCAFFOLD

Abstract. The selection of novel proteins or enzymes from random protein libraries has come to be a major objective in current biology, and these enzymes should prove useful in various biological and biomedical fields. New technologies such as in vitro selection of proteins in cell-free systems have high potential to realize evolu tionary molecular engineering of proteins. This review highlights an application of insertional mutagenesis of proteins to evolutionary molecular engineering. Random sequence proteins are inserted into the surface of a host enzyme which serves as a scaffold to display random protein libraries. Constraints on random polypeptide conformations owing to the proximity of N- and C-termini on the scaffold would result in greater screening efficiency of libraries. The scaffold enzyme is also used as a probe for monitoring the hill climbing of random sequence proteins on a fitness landscape and navigating rapid protein folding in the sequence space.