A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.

The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-β protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors.

A lipid transfer protein ensures nematode cuticular impermeability.

The cuticle of is impermeable to chemicals, toxins, and pathogens. However, increased permeability is a desirable phenotype because it facilitates chemical uptake. Surface lipids contribute to the permeability barrier.

HR-Bac, a toolbox based on homologous recombination for expression, screening and production of multiprotein complexes using the baculovirus expression system.

The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested.

Insect Cells-Baculovirus System for the Production of Difficult to Express Proteins: From Expression Screening for Soluble Constructs to Protein Quality Control.

Rapid preparation of proteins for functional and structural analysis is a major challenge both in academia and industry. The number potential targets continuously increases and many are difficult to express proteins which, when produced in bacteria, result in insoluble and/or misfolded recombinant proteins, protein aggregates, or unusable low protein yield. We focus here on the baculovirus expression vector system which is now commonly used for heterologous production of human targets.

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX.

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method.

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins.

Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors.

Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines.

Production of Multiprotein Complexes Using the Baculovirus Expression System: Homology-Based and Restriction-Free Cloning Strategies for Construct Design.

Most cellular processes are mediated by multi-subunit protein complexes which have attracted major interest in both academia and industry. Recombinant production of such entities in quantity and quality sufficient for functional and structural investigations may be extremely challenging and necessitate specific technologies. The baculovirus expression vector system is widely used for the production of eukaryotic multiprotein complexes, and a variety of strategies are available to assemble transfer vectors for the generation of recombinant baculoviruses.

In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair.

The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction.

Identification and characterization of the Onchocerca volvulus Excretory Secretory Product Ov28CRP, a putative GM2 activator protein.

Onchocerca volvulus is the nematode pathogen responsible for human onchocerciasis also known as "River blindness", a neglected tropical disease that affects up to 18 million people worldwide. Helminths Excretory Secretory Products (ESPs) constitute a rich repertoire of molecules that can be exploited for host-parasite relationship, diagnosis and vaccine studies. Here, we report, using a range of molecular techniques including PCR, western blot, recombinant DNA technology, ELISA, high performance thin-layer chromatography and mass spectrometry that the 28 KDa cysteine-rich protein (Ov28CRP) is a reliable component of the O.

Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression.

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation.

TFIIH: A multi-subunit complex at the cross-roads of transcription and DNA repair.

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both eukaryotic transcription and DNA repair, revealing a tight connection between these two processes. Composed of 10 subunits, it can be resolved into a 7-subunits core complex with the XPB translocase and the XPD helicase, and the 3-subunits kinase complex CAK, which also exists as a free complex with a distinct function. Initially identified as basal transcription factor, TFIIH also participates in transcription regulation and plays a key role in nucleotide excision repair (NER) for opening DNA at damaged sites, lesion verification and recruitment of additional repair factors.

The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH.

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens.

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP 2-K) is an enzyme that catalyses the formation of phytic acid (IP) from IP and ATP. In mammals, IP is involved in multiple events such as DNA repair and mRNA edit and it is the precursor of inositol pyrophosphates, emerging compounds shown to have an essential role in apoptosis. In addition, IP 2-K have functions in cells independently of its catalytic activity, for example in rRNA biogenesis.

An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb.

The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T.

Baculovirus expression: old dog, new tricks.

Since its inception more than 30 years ago, the baculovirus expression vector system (BEVS) has been used prolifically to produce heterologous proteins for research and development. In the cell, a cornerstone of biological activity are multiprotein complexes, catalyzing essential functions. BEVS has been uniquely successful to unlock such complex assemblies for high-resolution structural and functional analysis.

The production of multiprotein complexes in insect cells using the baculovirus expression system.

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck.

Insect cells-baculovirus system for the production of difficult to express proteins.

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system.

In TFIIH, XPD helicase is exclusively devoted to DNA repair.

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair.

ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities.

The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the "ARCH domain." By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex.

Expression of functional full-length hSRC-1 in eukaryotic cells using modified vaccinia virus Ankara and baculovirus.

Purified protein expression level and quality are contingent upon specific host expression systems. This differential production is particularly observed for proteins of high molecular weight, hampering further structural studies. We developed an expression method aimed at producing proteins in Escherichia coli, insect, and mammalian systems.