Facile Construction of Random Gene Mutagenesis Library for Directed Evolution Without the Use of Restriction Enzyme in Escherichia coli

A foolproof protocol was developed for the construction of mutant DNA library for directed protein evolution. First, a library of linear mutant gene was generated by error‐prone PCR or molecular shuffling, and a linear vector backbone was prepared by high‐fidelity PCR. Second, the amplified insert and vector fragments were assembled by overlap‐extension PCR with a pair of 5'‐phosphorylated primers. Third, full‐length linear plasmids with phosphorylated 5'‐ends were self‐ligated with T4 ligase, yielding circular plasmids encoding mutant variants suitable for high‐efficiency transformation. Self‐made competent Escherichia coli BL21(DE3) showed a transformation efficiency of 2.4 × 10⁵ cfu/µg of the self‐ligated circular plasmid. Using this method, three mutants of mCherry fluorescent protein were found to alter their colors and fluorescent intensities under visible and UV lights, respectively. Also, one mutant of 6‐phosphorogluconate dehydrogenase from a thermophilic bacterium Moorella thermoacetica was found to show the 3.5‐fold improved catalytic efficiency (k_cat/K_m) on NAD⁺ as compared to the wild‐type. This protocol is DNA‐sequence independent, and does not require restriction enzymes, special E. coli host, or labor‐intensive optimization. In addition, this protocol can be used for subcloning the relatively long DNA sequences into any position of plasmids.     

Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries

Successful antibody development requires both functional binding and desirable biophysical characteristics. In the current study, we analyze the causes of one hurdle to clinical development, off-target reactivity, or nonspecificity. We used a high-throughput nonspecificity assay to isolate panels of nonspecific antibodies from two synthetic single-chain variable fragment libraries expressed on the surface of yeast, identifying both individual amino acids and motifs within the complementarity-determining regions which contribute to the phenotype. We find enrichment of glycine, valine, and arginine as both individual amino acids and as a part of motifs, and additionally enrichment of motifs containing tryptophan. Insertion of any of these motifs into the complementarity-determining region H3 of a “clean” antibody increased its nonspecificity, with greatest increases in antibodies containing Trp or Val motifs. We next applied these rules to the creation of a synthetic diversity library based on natural frameworks with significantly decreased incorporation of such motifs and demonstrated its ability to isolate binders to a wide panel of antigens. This work both provides a greater understanding of the drivers of nonspecificity and provides design rules to increase efficiency in the isolation of antibodies with drug-like properties.     

Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17)

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn²⁺-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on neutrophil collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.     

Identification and Characterization of an Inhibitor of Trichothecene 3-O-Acetyltransferase,TRI101, by the Chemical Array Approach

Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.     

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.     

A Judgment on Postmortem Aging in Longissimus Dorsi Based on a Peptide Substrate Library

We attempted to develop a method to determine easily and effectively the degree of postmortem aging of pork longissimus dorsi (LD) by measuring the activity of proteases in the LD using fluorogenic peptide substrates. LD was used to measure the change with time in the protease activity detected with these substrates. Determining the variations within the LD muscles, strong positive correlations were found between changes in hardness and fluorescence intensities against Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.005), and strong negative correlations were found between changes in total amounts of free amino acids and Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P<0.001). Negative correlations were also observed between changes in the amounts of free Ala, Arg, Lys, Leu, Met, Phe, and Tyr and the fluorescence intensities against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA respectively (P<0.001).     

Synthetic approach to the generation of antibody diversity

The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494]     

A global metagenomic map of urban microbiomes and antimicrobial resistance

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