Directed Evolution Methods for Enzyme Engineering.

Enzymes underpin the processes required for most biotransformations. However, natural enzymes are often not optimal for biotechnological uses and must be engineered for improved activity, specificity and stability. A rich and growing variety of wet-lab methods have been developed by researchers over decades to accomplish this goal. In this review such methods and their specific attributes are examined.

Going native: Complete removal of protein purification affinity tags by simple modification of existing tags and proteases.

Protein purification typically involves expressing a recombinant gene comprising a target protein fused to a suitable affinity tag. After purification, it is often desirable to remove the affinity tag to prevent interference with downstream functions of the target protein. This is mainly accomplished by placing a protease site between the tag and the target protein. Typically, a small oligopeptide 'stub' C-terminal to the cleavage site remains attached to the target protein due to the requirements of sequence-specific proteases. Furthermore, steric hindrance can also limit protease efficiency. Here, we show that respectively fusing the interacting ePDZ-b/ARVCF protein-peptide pair to the target protein and a protease enables efficient processing of a minimised sequence comprising only residues N-terminal to the cleavage site. Interaction of the protein-peptide pair enforces proximity of the protease and its minimised cleavage sequence, enhancing both catalysis of a sub-optimal site and overcoming steric hindrance. This facilitates the high yield purification of fully native target proteins without recourse to specialised purification columns.

Compartmentalized linkage of genes encoding interacting protein pairs.

Emulsion technology has been successfully applied to the fields of next-generation high-throughput sequencing, protein engineering and clinical diagnostics. Here, we extend its scope to proteomics research by developing and characterizing a method, termed iCLIP (in vitro compartmentalized linkage of interacting partners), which enables genes encoding interacting protein pairs to be linked in a single segment of DNA. This will facilitate archiving of the interactomes from library versus library two-hybrid screens as libraries of linked DNAs. We further demonstrate the ability to interrogate a model yeast two-hybrid iCLIP library for interactants by "PCR-pulldown," using a primer specific to a gene of interest along with a universal primer. iCLIP libraries may also be subjected to high-throughput sequencing to generate interactome information. The applicability of the technique is also demonstrated in the related context of the bacterial two-hybrid system.

Novel Modalities in DNA Data Storage.

The field of storing information in DNA has expanded exponentially. Most common modalities involve encoding information from bits into synthesized nucleotides, storage in liquid or dry media, and decoding via sequencing. However, limitations to this paradigm include the cost of DNA synthesis and sequencing, along with low throughput. Further unresolved questions include the appropriate media of storage and the scalability of such approaches for commercial viability. In this review, we examine various storage modalities involving the use of DNA from a systems-level perspective. We compare novel methods that draw inspiration from molecular biology techniques that have been devised to overcome the difficulties posed by standard workflows and conceptualize potential applications that can arise from these advances.

Evolving a Thermostable Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of a 3' reversibly blocked nucleotide, followed by deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3' modified nucleotides, and TdT engineering is hampered by the fact that TdT is marginally stable and only present in mesophilic organisms. We sought to first evolve a thermostable TdT variant to serve as backbone for subsequent evolution to enable efficient incorporation of 3'-modified nucleotides. A thermostable variant would be a good starting point for such an effort, as evolution to incorporate bulky modified nucleotides generally results in lowered stability. In addition, a thermostable TdT would also be useful when blunt dsDNA is a substrate as higher temperature could be used to melt dsDNA. Here, we developed an assay to identify thermostable TdT variants. After screening about 10 000 TdT mutants, we identified a variant, named TdT3-2, that is 10 °C more thermostable than WT TdT, while preserving the catalytic properties of the WT enzyme.

Protein and Protease Sensing by Allosteric Derepression.

Peptide motifs are crucial mediators of protein-protein interactions as well as sites of specific protease activity. The detection and characterization of these events is therefore indispensable for a detailed understanding of cellular regulation. Here, we present versatile and modular sensors that allow the user to detect protease activity and protein-peptide interactions, as well as to screen for inhibitors using chromogenic, fluorescent, or luminescent output.

Rapid screening of protein-protein interaction inhibitors using the protease exclusion assay.

We have previously developed a sensitive and modular homogenous biosensor system using peptides to detect target ligands. By transposing the basic mechanistic principle of the nuclease protection assay into this biosensor framework, we have developed the protease exclusion (PE) assay which can discern antagonists of protein-protein interactions in a rapid, single-step format. We demonstrate the concept with multiple protein-peptide pairs and validate the method by successfully screening a small molecule library for compounds capable of inhibiting the therapeutically relevant p53-Mdm2 interaction. The Protease Exclusion method adds to the compendium of assays available for rapid analyte detection and is particularly suited for drug screening applications.

A generic scaffold for conversion of peptide ligands into homogenous biosensors.

Numerous peptide ligands including protease recognition sequences, peptides mediating protein-protein interactions, peptide epitopes of antibodies and mimotopes are available which bind molecules of interest. However, there is currently no facile method for the incorporation of these peptides into homogenous detection systems. We present a generalizable method for the incorporation of such peptides into a novel fusion protein framework comprising an enzyme and its inhibitor. The incorporated peptide functions as an allosteric hinge, linking enzyme to its inhibitor. Upon interaction with its cognate analyte, the peptide mediates dissociation of the inhibitor from the enzyme, and facilitates one-step signal generation. Likewise, cleavage of the peptide by a specific protease also causes enzyme-inhibitor dissociation, leading to signal generation. Using the ß-lactamase Tem1 and its inhibitor protein as a model scaffold, we show both specific and sensitive (between low nanomolar and mid-picomolar) colorimetric detection of proteases and antibodies within minutes in a homogenous one-step reaction visible to the naked eye. The same scaffold affords in vivo detection of antibody binding and protease function by linking activity to a selectable phenotype in E. coli.

DNA damage checkpoint maintains CDH1 in an active state to inhibit anaphase progression.

DNA damage checkpoint prevents segregation of damaged chromosomes by imposing cell-cycle arrest. In budding yeast, Mec1, Chk1, and Rad53 (homologous to human ATM/ATR, Chk1, and Chk2 kinases, respectively) are among the main effectors of this pathway. The DNA damage checkpoint is thought to inhibit chromosome segregation by preventing separase-mediated cleavage of cohesins. Here, we describe a regulatory network that prevents segregation of damaged chromosomes by restricting spindle elongation and acts in parallel with inhibition of cohesin cleavage. This control circuit involves Rad53, polo kinase, the anaphase-promoting complex activator Cdh1, and the bimC kinesin family proteins Cin8 and Kip1. The inhibition of polo kinase by Rad53-dependent phosphorylation prevents it from inactivating Cdh1. As a result, Cdh1 remains in a partially active state and limits Cin8 and Kip1 accumulation, thereby restraining spindle elongation. Hence, the DNA damage checkpoint suppresses both cohesin cleavage and spindle elongation to preserve chromosome stability.

Consorting kinases, end of destruction and birth of a spindle.

Centrosomes (spindle pole body in yeast) constitute the two poles of the bipolar mitotic spindle and play a prominent role in the segregation of chromosomes during mitosis. Like chromosomes, the centrosome inherited from the progenitor cell duplicates once in each division cycle, following which the sister centrosomes segregate away from each other to assemble a short spindle upon initiation of mitosis. Cdh1, an activator of the E3 ubiquitin ligase APC (Anaphase Promoting Complex), is a potent inhibitor of centrosome segregation and suppresses spindle assembly during S phase by mediating proteolytic destruction of the microtubule associated proteins (MAPs) required for centrosome separation. A recent study in yeast suggests that concerted action by two prominent kinases Cdk1 and polo are required to bring this destruction to a halt by inactivating Cdh1 and to facilitate spindle assembly. This is an effective strategy for the modulation of the activities of cell cycle regulators that require multiple phosphorylation. The control circuit involving Cdh1, Cdk1, Polo and MAPs may be also targeted by other cellular networks in contexts that demand the restraining of spindle dynamics.

DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior.

The DNA replication checkpoint maintains replication fork integrity and prevents chromosome segregation during replication stresses. Mec1 and Rad53 (human ATM/ATR- and Chk2-like kinases, respectively) are critical effectors of this pathway in yeast. When treated with replication inhibitors, checkpoint-deficient mec1 or rad53 mutant fails to maintain replication fork integrity and proceeds to partition unreplicated chromosomes. We show that this unnatural chromosome segregation requires neither the onset of mitosis nor APC activation, cohesin cleavage, or biorientation of kinetochores. Instead, the checkpoint deficiency leads to deregulation of microtubule-associated proteins Cin8 and Stu2, which, in the absence of both chromosome cohesion and bipolar attachment of kinetochores to microtubules, induce untimely spindle elongation, causing premature chromosome separation. The checkpoint's ability to prevent nuclear division is abolished by combined deficiency of microtubule-destabilizing motor Kip3 and Mad2 functions. Thus, the DNA replication checkpoint prevents precocious chromosome segregation, not by inhibiting entry into mitosis as widely believed, but by directly regulating spindle dynamics.