Transient Co-expression of Membrane Protein Complexes in Mammalian Cells.

Membrane proteins are essential components of biological membranes with key roles in cellular processes such as nutrient transport, cell communication, signaling, or energy conversion. Due to their crucial functions, membrane proteins and their complexes are often targets for therapeutic interventions. Expression and purification of membrane proteins are often a bottleneck to yield sufficient material for structural studies and further downstream characterization. Taking advantage of the Expi293 expression system for the production of eukaryotic proteins, we present a very efficient and fast protocol for the co-expression of a membrane complex. Here, we use transient transfection to co-express the membrane transporter PHT1 with its adaptor protein TASL. To allow the simultaneous screening of different proteins, constructs, or interaction partners, we make use of the Twin-Strep magnetic system. The protocol can be applied for small-scale screening of any membrane protein alone or co-expressed with interacting partners followed by large-scale production and purification of a potential membrane protein complex.

Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

Strategies for rapid production of crystallization quality coatomer WD40 domains.

The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and ß'-COPI subunits to bind these membrane protein "clients" for ER retrieval. These "αWD40" and "ß'WD40" domains are structural homologs that demonstrate distinct selectivity for client proteins. However, elucidation of the atomic-level principles of coatomer-client interactions has been challenging due to the tendency of αWD40 domain to undergo aggregation during expression and purification. Here we describe a rapid recombinant production strategy from E. coli, which substantially enhances the quality of the purified αWD40 domain. The αWD40 purification and crystallization are completed within one day, which minimizes aggregation losses and yields a 1.9 Å resolution crystal structure. We demonstrate the versatility of this strategy by applying it to purify the ß'WD40 domain, which yields crystal structures in the 1.2-1.3 Å resolution range. As an alternate recombinant production system, we develop a cost-effective strategy for αWD40 production in human Expi293 cells. Finally, we suggest a roadmap to simplify these protocols further, which is of significance for the production of WD40 mutants prone to rapid aggregation. The WD40 production strategies presented here are likely to have broad applications because the WD40 domain represents one of the largest families of biomolecular interaction modules in the eukaryotic proteome and is critical for trafficking of host as well as viral proteins such as the SARS-CoV-2 spike protein.

A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells.

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

Rapid generation of high-quality recombinant antibodies using an Expi293F expression system for a 17 ß-estradiol immunoassay.

The rapid generation of high-quality target antibodies is essential for research employing immunoassays. The use of recombinant antibody technology that relies on genetic engineering is one such means to produce high-quality antibodies. Obtaining the gene sequence information of immunoglobulin is a prerequisite for the preparation of genetically engineered antibodies. At present, many researchers have shared their amino acid sequence data for various high-performance antibodies and their related properties. In this study, we obtained the protein sequence of a variable region of a 17 ß-estradiol (E2) antibody from the Protein Data Bank (PDB) and subsequently constructed heavy (H) and light (L) chain expression vectors through codon optimization. The transient expression, purification, and performance identification of the immunoglobulin G (IgG), antigen-binding fragment (Fab), and single-chain variable fragment (scFv) antibodies were carried out, respectively. The effects of the different expression vectors on the expression yield of the IgG antibody were further compared. Among them, the expression yield based on the pTT5 vector was the highest, reaching 27 mg/L. Based on the expressed IgG and Fab antibodies, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) standard curve of E2 was constructed, and the half-maximal inhibitory concentrations (IC50) for these two antibodies were determined to be 0.129 ng/mL and 0.188 ng/mL, respectively. In addition, an immunochromatographic assay (ICA) based on the IgG antibody was constructed with an IC50 of 3.7 ng/mL. Therefore, in featuring the advantages of simplicity, high efficiency, rapid obtainment, and high titer yield, we propose the system for the rapid generation of high-quality recombinant antibodies by reusing the published antibody information and show that it has good implementation prospects in improving upon existing immunoassay techniques.

Development of an efficient method for selection of stable cell pools for protein expression and surface display with Expi293F cells.

Compare with transient expression, stable cell lines generally have higher productivity and better quality for protein expression. However, selection of stable cell line is time-consuming and laborious. Here we describe an optimized selection method to achieve high-efficient stable cell pools with Expi293F suspension cells. This method only takes 2-3 weeks to generate stable cell pools with 2- to 10-fold higher productivity than transient gene expression (TGE). In fed-batch culture with Yeastolate, >1 g/L yield was achieved with our KTN0239-IgG stable cell pool in shaker flasks. This method can be also applied to efficiently display proteins on the cell surface.

Novel Protein Production Method Combining Native Expression in Human Cells with an Intein-based Affinity Purification and Self-cleavable Tag.

The human proteins used in most biochemical studies are commonly obtained using bacterial expression. Owing to its relative simplicity and low cost, this approach has been extremely successful, but is inadequate for many proteins that require the mammalian folding machinery and posttranslational modifications (PTMs) for function. Moreover, the expressed proteins are typically purified using N- and/or C-terminal affinity tags, which are often left on proteins or leave non-native extra amino acids when removed proteolytically. Many proteins cannot tolerate such extra amino acids for function. Here we describe a protein production method that resolves both these issues. Our method combines expression in human Expi293F cells, which grow in suspension to high density and can process native PTMs, with a chitin-binding domain (CBD)-intein affinity purification and self-cleavable tag, which can be precisely removed after purification. In this protocol, we describe how to clone a target gene into our specifically designed human cell expression vector (pJCX4), and how to efficiently transfect the Expi293F cells and purify the expressed proteins using a chitin affinity resin. Graphic abstract.

Transient Gene Expression in Human Expi293 Cells.

Transient gene expression (TGE) is an important tool for generating recombinant proteins in a short period of time. The human cell line HEK293 is widely used for this purpose since it can grow in suspension to a high cell density in serum-free media. In addition, this cell line is amenable to several transfection methods and produces recombinant proteins in satisfactory quantities for functional and structural analysis. This chapter describes the methodology for TGE using the Expi293 system, which provides higher expression levels than other HEK293-based systems.

Transient Transfection and Expression of Eukaryotic Membrane Proteins in Expi293F Cells and Their Screening on a Small Scale: Application for Structural Studies.

Cancers, neurodegenerative and infectious diseases remain some of the leading causes of deaths worldwide. The structure-guided drug design is essential to advance drug development for these important diseases. One of the key challenges in the structure determination workflow is the production of eukaryotic membrane proteins (drug targets) of high quality. A number of expression systems have been developed for the production of eukaryotic membrane proteins. In this chapter, an optimized detailed protocol for transient transfection and expression of eukaryotic membrane proteins in Expi293F cells is presented. Testing expression and purification on a small scale allow optimizing conditions for sample preparation for downstream structural (cryo-EM) elucidation.

Purification of the Dynamin-Related Protein Vps1 Using Mammalian and Bacterial Expression Systems.

The dynamin-related proteins (DRPs) are self-assembling membrane remodeling machines that are indispensable for fundamental cellular trafficking and homeostatic processes. We describe in this chapter protocols developed in our laboratory for purification of full-length and minimal constructs of Chaetomium thermophilum Vps1, the model fungal DRP, using mammalian and Escherichia coli expression systems.

Expression and Preparation of Recombinant Reelin and ADAMTS-3 Proteins.

Reelin is a large secreted protein that is essential for the brain development and function. Reelin is negatively regulated by the specific cleavage by a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3) which is also secreted from neurons. It is likely that there are other proteases that can cleave Reelin. This chapter describes the protocol for expression and handling of recombinant Reelin and ADAMTS-3 proteins to facilitate investigation of these proteins.

Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells.

BACKGROUND: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.. METHODS: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost). RESULTS: The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aß) aggregates in vivo. CONCLUSIONS: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers.

Generation of recombinant destabilase-lysozyme from medicinal leeches in three different expression systems.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.