Applications of Golden Gate cloning to protein production using the baculovirus expression vector system.

Advances in structural biology techniques over the last decades have made it increasingly possible to determine the structures of multi-protein complexes. Generation of sufficient recombinant material for such studies remains a bottleneck and often requires screening a variety of purification strategies and different subunit compositions to reproducibly isolate homogeneous complexes. Parallel advances in molecular biology now make it possible to easily generate panels of constructs with different affinity tags and different multi-protein components. Here, we describe two protocols based on Golden Gate cloning, which facilitate the generation of multi-protein complexes for protein production via the Baculovirus Expression Vector System. This robust method makes it possible to efficiently generate a panel of multi-gene expression constructs containing up to 15 open reading frames.

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.

GoldenBac: a simple, highly efficient, and widely applicable system for construction of multi-gene expression vectors for use with the baculovirus expression vector system.

BACKGROUND: Recombinant protein production and purification of large protein complexes in eukaryotes requires efficient methods to generate multi-gene expression constructs, where each individual gene is under the control of its own promoter and terminator. Current methods are based either on serial rounds of combination of several vectors containing loxP sites via the Cre-lox technology, or on multiple rounds of gene combination via PCR or other methods. These methods are multi-step, have lower efficiencies than single gene cloning, and may require laborious processes to verify that all genes of interest are present in the final product. Here, we describe a rapid and simple Golden Gate-based system for the generation of multi-gene expression constructs compatible with baculovirus expression vector systems (BEVS) using either Tn7 transposition or KO1629-based homologous recombination, which we refer to as "GoldenBac". RESULTS: This method is based on the construction of a series of vectors containing a promoter-gene of interest-terminator cassette flanked by cleavage sites of the BsaI type IIS restriction enzyme. This series of vectors can be cut by BsaI to excise cassettes with unique overhangs. In the same reaction, the cassettes are then ligated in the correct sequence in a final destination vector to generate multi-gene expression constructs containing 2-15 genes. Individual expression constructs can therefore be combined into a single vector in a single reaction, with over 90% efficiency when combining up to 14 expression cassettes. We demonstrate successful construction and expression of three different co-expression systems, the proteosomal lid complex, the anaphase promoting complex/cyclosome (APC/C), and a series of constructs used to test the effect of chaperone co-expression on the solubility of the HOIP protein. CONCLUSIONS: This robust, single-step cloning system provides an easy-to-use method for generation of multi-gene expression constructs for both transposition and homologous recombination-based baculovirus systems, making this technology available across all laboratories using baculovirus expression systems. This highly efficient and simple method allows for rapid incorporation of multi-gene expression cloning into the standardized service portfolio of protein production facilities and can also easily be adopted by any laboratory for routine generation of multi-gene baculovirus constructs.

Map of physical interactions between extracellular domains of Arabidopsis leucine-rich repeat receptor kinases.

Plants use surface receptors to perceive information about many aspects of their local environment. These receptors physically interact to form both steady state and signalling competent complexes. The signalling events downstream of receptor activation impact both plant developmental and immune responses. Here, we present a comprehensive study of the physical interactions between the extracellular domains of leucine-rich repeat receptor kinases (LRR-RKs) in Arabidopsis. Using a sensitized assay, we tested reciprocal interactions among 200 of the 225 Arabidopsis LRR-RKs for a total search space of 40,000 interactions. Applying a stringent statistical cut-off and requiring that interactions performed well in both bait-prey and prey-bait orientations resulted in a high-confidence set of 567 bidirectional interactions. Additionally, we identified a total of 2,586 unidirectional interactions, which passed our stringent statistical cut-off in only one orientation. These datasets will guide further investigation into the regulatory roles of LRR-RKs in plant developmental and immune signalling decisions.

Multiplex mutagenesis of four clustered CrRLK1L with CRISPR/Cas9 exposes their growth regulatory roles in response to metal ions.

Resolving functions of closely linked genes is challenging or nearly impossible with classical genetic tools. Four members of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) family are clustered on Arabidopsis chromosome five. To resolve the potentially redundant functions of this subclass of CrRLK1Ls named MEDOS1 to 4 (MDS1 to 4), we generated a single CRISPR/Cas9 transformation vector using a Golden Gate based cloning system to target all four genes simultaneously. We introduce single mutations within and deletions between MDS genes as well as knock-outs of the whole 11 kb gene cluster. The large MDS cluster deletion was inherited in up to 25% of plants lacking the CRISPR/Cas9 construct in the T2 generation. In contrast to described phenotypes of already characterized CrRLK1L mutants, quadruple mds knock-outs were fully fertile, developed normal root hairs and trichomes and responded to pharmacological inhibition of cellulose biosynthesis similar to wildtype. Recently, we demonstrated the role of four CrRLK1L in growth adaptation to metal ion stress. Here we show the involvement of MDS genes in response to Ni2+ during hypocotyl elongation and to Cd2+ and Zn2+ during root growth. Our finding supports the model of an organ specific network of positively and negatively acting CrRLK1Ls.

Publisher Correction: An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

In this Letter, an incorrect version of the Supplementary Information file was inadvertently used, which contained several errors. The details of references 59-65 were missing from the end of the Supplementary Discussion section on page 4. In addition, the section 'Text 3. Y2H on ICD interactions' incorrectly referred to 'Extended Data Fig. 4d' instead of 'Extended Data Fig. 3d' on page 3. Finally, the section 'Text 4. Interaction network analysis' incorrectly referred to 'Fig. 1b and Extended Data Fig. 6' instead of 'Fig. 2b and Extended Data Fig. 7' on page 3. These errors have all been corrected in the Supplementary Information.

An extracellular network of Arabidopsis leucine-rich repeat receptor kinases.

The cells of multicellular organisms receive extracellular signals using surface receptors. The extracellular domains (ECDs) of cell surface receptors function as interaction platforms, and as regulatory modules of receptor activation. Understanding how interactions between ECDs produce signal-competent receptor complexes is challenging because of their low biochemical tractability. In plants, the discovery of ECD interactions is complicated by the massive expansion of receptor families, which creates tremendous potential for changeover in receptor interactions. The largest of these families in Arabidopsis thaliana consists of 225 evolutionarily related leucine-rich repeat receptor kinases (LRR-RKs), which function in the sensing of microorganisms, cell expansion, stomata development and stem-cell maintenance. Although the principles that govern LRR-RK signalling activation are emerging, the systems-level organization of this family of proteins is unknown. Here, to address this, we investigated 40,000 potential ECD interactions using a sensitized high-throughput interaction assay, and produced an LRR-based cell surface interaction network (CSILRR) that consists of 567 interactions. To demonstrate the power of CSILRR for detecting biologically relevant interactions, we predicted and validated the functions of uncharacterized LRR-RKs in plant growth and immunity. In addition, we show that CSILRR operates as a unified regulatory network in which the LRR-RKs most crucial for its overall structure are required to prevent the aberrant signalling of receptors that are several network-steps away. Thus, plants have evolved LRR-RK networks to process extracellular signals into carefully balanced responses.

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

Random mutagenesis of the prokaryotic peptide transporter YdgR identifies potential periplasmic gating residues.

The peptide transporter (PTR) family represents a group of proton-coupled secondary transporters responsible for bulk uptake of amino acids in the form of di- and tripeptides, an essential process employed across species ranging from bacteria to humans. To identify amino acids critical for peptide transport in a prokaryotic PTR member, we have screened a library of mutants of the Escherichia coli peptide transporter YdgR using a high-throughput substrate uptake assay. We have identified 35 single point mutations that result in a full or partial loss of transport activity. Additional analysis, including homology modeling based on the crystal structure of the Shewanella oneidensis peptide transporter PepT(so), identifies Glu(56) and Arg(305) as potential periplasmic gating residues. In addition to providing new insights into transport by members of the PTR family, these mutants provide valuable tools for further study of the mechanism of peptide transport.

Casein kinase I epsilon somatic mutations found in breast cancer cause overgrowth in Drosophila.

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.