Virus-like particles (VLPs) were produced in stable transgenic plants expressing hepatitis B surface antigen (HBsAg, Mason et al., 1992) and Norwalk virus capsid protein (NVCP, Mason et al., 1996). These studies led to transgenic potato lines expressing HBsAg or NVCP that were used in clinical trials, showing oral immunogenicity. In the past two decades, transient expression systems for plants using viral replicons were developed that enable robust production of recombinant proteins. These have been further optimized and result in very efficient production of hepatitis B core antigen (HBcAg) and Norovirus VLP.  HBcAg heterodimer VLPs were used for display of heterologous antigens, and when co-delivered with plant-made recombinant immune complexes, produced immune synergy in mice. 

Production of recombinant proteins in E. coli is the engine of many drug discovery efforts. Supplying these protein reagents can be the rate-limiting step. Another bacterial host, Vibrio natriegens, has recently shown promise as an alternative to E. coli. Here, we present a set of general protocols for its use. Moreover, we show that Vibrio natriegens can outproduce E. coli for certain protein reagents, and/or solubilize protein where E. coli fails.

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with specific bound peptides (pMHC-I) for research, diagnostic, and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone, TAP-binding protein-related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E. coli.  

The increasing demands for protein reagents needed for drug discovery efforts drive the development of an efficient methodology for recombinant protein production.  Within the BioMolecular Resources Department at Genentech, we have developed a platform for high throughput construct generation and expression screening in baculovirus, E coli and mammalian transient systems applicable to all protein types and localizations.

We will describe the development of E. coli SuptoxD and SuptoxR, two specialized strains for high-level recombinant membrane protein (MP) production. These engineered strains can: (1) suppress the toxicity that frequently accompanies MP overexpression, thus enabling enhanced levels of final bacterial biomass; and (2) markedly increase the cellular accumulation of membrane-embedded protein. Combined, these two positive effects result in dramatically enhanced volumetric yields for various prokaryotic and eukaryotic recombinant MPs.

Protein S-fatty acylation refers to the post-translational attachment of fatty acids (16-carbon palmitate being the most common) to proteins. In 2002, the identity of the enzymes that catalyze protein S-fatty acylation was revealed. Referred to as the DHHC palmitoyltransferases, these enzymes comprise a highly diverse family with 23 members in humans. The biomedical importance of the members of this family is underscored by its association with a variety of human diseases, including intellectual disability, Huntington’s Disease, schizophrenia, and cancer. Essential and unique roles for DHHC proteins have been identified using DHHC-deficient mouse models. Phenotypes observed include neurodevelopmental deficits, defective learning and memory, and neurodegeneration. Accordingly, there is significant interest in understanding the mechanism and regulation of DHHC enzymes. Biochemical and mechanistic studies on DHHC proteins remain challenged by the innate difficulty of purifying the enzyme in large scale. Standard bacterial expression systems lack the enzymes required for post-translational modification, and hence are not suited for purifying these eukaryotic membrane enzymes. Moreover, preservation of their biological and functional activities during the isolation process can be compromised. In this talk, I will describe a protocol we developed for successfully expressing and purifying recombinant DHHC proteins, using DHHC3 and its catalytically inactive cysteine mutant DHHS3 as examples. Homogeneity and monodispersity of the purified protein are examined by size exclusion chromatography.

Establishing monoclonality of research cell banks (RCB) and master cell banks (MCB) is critical for successful and cost-effective biologic manufacturing campaigns and regulatory filings. Using whole genome sequencing (WGS) and proprietary bioinformatics tools, Selexis is able to establish monoclonality through an unbiased approach that identifies and validates integration sites, establishes clonality using integration sites as markers and provide transgene sequence, copy number and integrity.

Multidrug resistance protein 4 (MRP4/ABCC4) can transport a range of organic anionic compounds out of the cell, and has been linked with cancer, inflammation and cell signalling. MRP4 was expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Subsequently MRP4 was solubilised and purified using novel detergents or polymers that conferred increased stability to the protein.

Monoclonal antibodies (mAbs) produced in CHO cells usually exhibit N-glycan patterns with only very little sialylation, rating this type of N-glycosylation rather uncritical within the CHO cell community. However, we have recently identified a CHO cell line producing mAbs with greater than 50% sialylation, while 30% actually accounted for NGNA sialylation. NGNA sialylation represents a critical quality attribute supposed to induce immunogenic reactions. In-depth transcriptomic and non-coding RNA expression analyses revealed a novel, putative, regulatory circuit around protein sialylation in CHO cells, which is driven by a newly identified small non-coding RNA. Our data suggest that specific changes in the CHO cell genome, such as mutations, can have a dramatic influence on the product quality of the produced therapeutic proteins.

Chinese hamster ovary (CHO) cells have been commonly used mammalian host cells for therapeutic protein production over the last 3 decades. As technology advances in CHO cell engineering and culturing, significant improvements have been achieved in protein production. Having worked with more than 150 recombinant proteins as a CDMO, KBI has observed and overcome a wide array of challenges with expression and purification.

Recombinant expression of GPCRs is difficult but important for developing assays, producing antigens for antibodies, and structural biology. We describe using cell-free expression with E. coli lysate to produce the G-protein coupled estrogen receptor (GPER). GPER was produced in microgram quantities, precipitated, and reconstituted. We describe the first use of recombinantly expressed GPER in a functional liquid chromatography-mass spectrometry assay to validate binding with known ligands.

Using Sorrento’s G-MAB™ library²⁶, a single chain variable fragment (scFv) antibody phage display library , we identified STI-1499, a neutralizing antibody that  binds the SARS-CoV-2 Spike S1 subunit. STI-1499 inhibits cytopathic effects of infection by genetically diverse clinical SARS-CoV-2 pandemic isolates in vitro, and has demonstrated efficacy in a hamster model of COVID-19 when administered by the intravenous route immediately following infection. Following affinity maturation of STI-1499, an antibody termed STI-2020 was isolated and characterized.  STI-2020 had demonstrated potent neutralizing activity following IV and IN administration up to 12 hours following infection in the hamster model.  Early clinical development of STI-1499 and STI-2020 is currently underway.