High-throughput Protein Production Research Articles

ESTABLISHMENT OF AN ULTRA HIGH-THROUGHPUT PROTEIN PRODUCTION PLATFORM (ULTRA 96) FOR EARLY STAGE IN VITRO STUDIES

Abstract Proteins are known as the building blocks of life which play many critical roles in the body. In early stage drug discovery, numerous μg to mg scale high quality proteins need to be produced for in vitro functional studies and therapeutic evaluation, including antigens, monoclonal antibodies (mAb), bispecific antibodies (bsAbs) as well as other Fc fusion proteins. High-throughput production (HTP) of various proteins with high quality in very short time is a big challenge in industry. Here we present how WuXi Biologics has created a powerful ultra HTP protein production platform (termed as “Ultra 96”) that can automatically produce proteins within 3 weeks with one step purification at 1~3mL expression scale by using 96 and 24 well DWP. High titer of expression, high quality of products, short timeline as well as low cost make “Ultra 96” a robust platform which can empower our clients and dramatically accelerate their early stage drug discovery projects.

Adaptation of the binding domain of Lactobacillus acidophilus S-layer protein as a molecular tag for affinity chromatography development.

The S-layer proteins are a class of self-assembling proteins that form bi-dimensional lattices named S-Layer on the cell surface of bacteria and archaea. The protein SlpA, which is the major constituent of the Lactobacillus acidophilus S-layer, contains in its C-terminus region (SlpA284 - 444), a protein domain (named here as SLAPTAG) responsible for the association of SlpA to the bacterial surface. SLAPTAG was adapted for the development of a novel affinity chromatography method: the SLAPTAG-based affinity chromatography (SAC). Proteins with different molecular weights or biochemical functions were fused in-frame to the SLAPTAG and efficiently purified by a Bacillus subtilis-derived affinity matrix (named Bio-Matrix or BM). Different binding and elution conditions were evaluated to establish an optimized protocol. The binding equilibrium between SLAPTAG and BM was reached after a few minutes of incubation at 4°C, with an apparent dissociation constant (KD) of 4.3μM. A reporter protein (H6-GFP-SLAPTAG) was used to compare SAC protein purification efficiency against commercial immobilized metal affinity chromatography. No differences in protein purification performance were observed between the two methods. The stability and reusability of the BM were evaluated, and it was found that the matrix remained stable for more than a year. BM could be reused up to five times without a significant loss in performance. Additionally, the recovery of bound SLAP-tagged proteins was explored using proteolysis with a SLAP-tagged version of the HRV-3c protease (SLAPASE). This released the untagged GFP while the cut SLAPTAG and the SLAPASE were retained in the BM. As an alternative, iron nanoparticles were linked to the BM, resulting in BMmag. The BMmag was successfully adapted for a magnetic SAC, a technique with potential applications in high-throughput protein production and purification. The SAC protocol can be adapted as a universal tool for the purification of recombinant proteins. Furthermore, the SAC protocol utilizes simple and low-cost reagents, making it suitable for in-house protein purification systems in laboratories worldwide. This enables the production of pure recombinant proteins for research, diagnosis, and the food industry.

Abstract 5323: Accelerating the drug discovery process with an automated high-throughput protein production and characterization platform for AI-driven antibody development of immunotherapy

Abstract The demonstrated effectiveness and vast potential of large molecule therapeutics have driven biopharmaceutical companies to change the ways by which they discover and develop novel biologic therapies. While the conventional antibody discovery process via animal immunization and in vivo hypermutation can generate sufficient hits for functional screening, this approach can only cover a limited subset of sequence diversity. In silico, artificial intelligence (AI) driven methods, by contrast, have the potential to cover 2-3 orders of magnitude more sequence diversity compared to conventional methods and more rapidly produce a drug candidate with a superior safety and efficacy profile. However, this approach requires wet lab validation of predicted sequences as well as targeted data generation to improve AI-predicted rankings. Traditional wet lab methods for protein generation and characterization are expensive, time consuming, and prone to human error. These limitations restrict the overall potential benefit of the AI-driven antibody discovery process. To accelerate this process, which consists of a complex series of cycles spanning different functional teams, a substantial increase in the throughput of the end-to-end production and characterization workflow is established in-house such that 104 antibodies with property data can be generated and analyzed within a short period of time. BioMap has implemented a highly integrated and automated protein expression, purification, and characterization platform linked with a unified informatics system to eliminate as much manual operation as possible. This high-throughput robotic platform interweaves multiple procedures including plasmid preparation, cell dispensing, mammalian transfection, protein purification, and characterization, and it is coupled to our in-house informatics system and database. Empowered by state-of-the-art liquid handling system and well-established experimental protocols, the integrated facility is capable of the delivery of 103 protein samples from plasmids in ten days within a single batch. Benefiting from our robust transient expression platform, the production scale ranges from 0.5 to 30 mL with an average antibody yield of 300 mg/L. The high-throughput workflow brings a challenge to track the provenance of each protein and manage data flow across different stations. To address this bottleneck, a well-defined informatics platform and database have been developed to provide sample registration, interface with lab instruments, experimental process tracking, and automated data recording and analysis. The implementation of this system provides a massive amount of high-quality data available for AI training and validation with a very short turnover time and enables AI-driven development of next-generation biologics. Citation Format: Zhehao Xiong, Wei Jiang, Lijun Xia, Jianhua Huang, Cheng-chi Chao. Accelerating the drug discovery process with an automated high-throughput protein production and characterization platform for AI-driven antibody development of immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5323.

The high-throughput production of membrane proteins.

Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.

Abstract 3253: A high-throughput platform to produce neoE-HLA libraries for capturing neoE-specific T cells from the peripheral blood of patients with solid tumors

Abstract A critical step in the development of personalized neoTCR-T cell therapies is operational selection of tumor-exclusive neoantigen peptide targets (neoepitopes or neoE) presented by each person's own HLA proteins (neoE-HLA targets). We have developed a high-throughput, automated process for the cloning, expression and purification of soluble proteins comprising neoE peptide with beta2m and the HLA heavy chain fused into a single polypeptide (neoE-HLA proteins). Of the 13 thousand HLA alleles in the human population, with 6 HLA alleles expressed in each person, our HLA catalog enables rapid production of neoE-HLA protein candidate libraries, representing >4 of 6 HLAs for ≥95% of all patients, regardless of ethnicity. We apply our proprietary bioinformatics pipeline to predict and prioritize tumor-exclusive neoE-HLA candidates for each patient. We then generate neoE-HLA libraries in single production runs for up to several hundred neoE-HLA candidates per patient. These soluble neoE-HLA proteins are then assembled into barcoded snare multimers. These barcoded snare libraries are used to interrogate matched PBMCs from that patient to capture rare (>1 per 106) to abundant antigen-experienced CD8 T cells which specifically bind the cognate neoE-HLA tumor targets, using the imPACT Isolation Technology®. This platform is capable of producing barcoded snares predicted to represent high affinity (≤500 nM) and low affinity (>500nM) neoE binding to their cognate HLAs. These capabilities permit deep and broad interrogation of the tumor-targeted neoE-specific T cell repertoire in the blood of patients with solid cancers. Data shown here demonstrate that >85% of patients with different cancer types, indeed, harbor a surprising repertoire of tumor-targeted neoE-specific T cells. These antigen-experienced T cells reveal the pre-existing immune response to each person's cancer, and how that repertoire evolves in longitudinal analyses of patients that respond/do not respond to their cancer therapies. In summary, PACTs proprietary neoE-HLA prediction pipeline and the proprietary high-throughput neoE-HLA protein production platform enables the discovery of tumor-targeted, neoE-specific T cells from patients. We leverage the neoTCRs cloned from these captured T cells to engineer fresh T cells from the patient into a full dose of tumor-targeted personalized neoTCR-T cells (NeoTCR-P1 T cell therapy). This fully personalized NeoTCR-P1 cell therapy is manufactured in fully enclosed, automatable systems for each individual patient with solid tumors. These breakthrough technologies support the on-going Phase 1 clinical trial of personalized engineered autologous NeoTCR-P1 T cell therapies for patients with six different solid tumor types (NCT03970382). Citation Format: Olivier Dalmas, Zheng Pan, Christine Shieh, Allison Xu, Jason Kwa, Katharine Heeringa, Yan Ma, John Collins, Duo An, Songming Peng, Alex Franzusoff. A high-throughput platform to produce neoE-HLA libraries for capturing neoE-specific T cells from the peripheral blood of patients with solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3253.

High throughput generation of a resource of the human secretome in mammalian cells

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as ‘secreted’ in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic β-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.

Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale

Cultivation of yeast Pichia pastoris in the microtiter plate, for optimisation of culture conditions, and expression screening of transformants has gained significance in recent years. However, in the microtiter plate, it has been challenging to attain cell densities similar to well-aerated shake-flask culture, due to the poor mixing resulting in oxygen limitation. To solve this problem, we investigated the influence of multiple cultivation parameters on P. pastoris cell growth, including the architecture of 96-deepwell plate (96-DWP), shaking throw diameter, shaking frequency, culture volume/well, and media composition. In the optimised conditions, a cell density of OD600 ~50 (dry cell weight ~13 g/L) with >99% cell viability was achieved in the casamino acids supplemented buffered-minimal-media in 300 to 1000 μl culture volume/well. We have devised a simplified method for coating of the culture supernatant on the polystyrene surface for immunoassay. Clones for secretory expression of envelope domain III of dengue virus serotype-1 under the control of inducible and constitutive promoter were screened using the developed method. Described microscale cultivation strategy can be used for rapid high-throughput screening of P. pastoris clones, media optimization, and high-throughput recombinant protein production. The knowledge gained through this work may also be applied, to other suspension cultures, with some modifications.

Crystal structure of Nsp15 endoribonuclease NendoU from SARS-CoV-2.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS‐CoV‐2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS‐CoVs and Middle East Respiratory Syndrome coronavirus (MERS‐CoVs), the detailed information about SARS‐CoV‐2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high‐throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS‐CoV‐2 proteins and structures. Here we report two high‐resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.

Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems.

Cell-free protein synthesis (CFPS) system is a simple, rapid, and sensitive tool that is devoid of membrane-bound barriers, yet contains all the mandatory substrates, biomolecules, and machineries required for the synthesis of the desired proteins. It has the potential to overcome loopholes in the current in vivo production systems and is a promising tool in both basic and applied scientific research. It facilitates a simplified organization of desired experiments with a variety of reaction conditions, making CFPS a powerful tool in biological research. It has been used for the expansion of genetic code, assembly of viruses, and in metabolic engineering for production of toxic and complex proteins. Subsequently, CFPS systems have emerged as potent technology for high-throughput production of membrane proteins, enzymes, and therapeutics. The present review highlights the recent advances and uses of CFPS systems in biomedical, therapeutic, and biotechnological applications. Additionally, we highlight possible solutions to the potential biosafety issues that may be encountered while using CFPS technology.

High-Throughput Protein Production Combined with High- Throughput SELEX Identifies an Extensive Atlas of Ciona robusta Transcription Factor DNA-Binding Specificities.

Transcription factors (TFs) control gene transcription, binding to specific DNA motifs located in cis-regulatory elements across the genome. The identification of TF-binding motifs is thus an important aspect to understand the role of TFs in gene regulation. SELEX, Systematic Evolution of Ligands by EXponential enrichment, is an efficient in vitro method, which can be used to determine the DNA-binding specificity of TFs. Thanks to the development of high-throughput (HT) DNA cloning system and protein production technology, the classical SELEX assay has be extended to high-throughput scale (HT-SELEX).We report here the detailed protocol for the cloning, production, and purification of 420 Ciona robusta DNA BD. 263 Ciona robusta TF DNA-binding domain proteins were purified in milligram quantities and analyzed by HT-SELEX. The identification of 139 recognition sequences generates an atlas of protein-DNA-binding specificities that is crucial for the understanding of the gene regulatory network (GRN) of Ciona robusta. Overall, our analysis suggests that the Ciona robusta repertoire of sequence-specific transcription factors comprises less than 500 genes. The protocols for high-throughput protein production and HT-SELEX described in this article for the study of Ciona robusta TF DNA-binding specificity are generic and have been successfully applied to a wide range of TFs from other species, including human, mouse, and Drosophila.

High-Throughput Protein Production in Yeast.

Yeasts are versatile single-celled fungi that grow to high cell densities on inexpensive media. With well-studied genetics and metabolism and a wealth of knowledge available about their propagation and growth in academic as well as industrial settings, yeasts have long been used for recombinant protein production of isolated proteins and multisubunit complexes. They can be easily adapted to high-throughput protein expression pipelines. Importantly, the outcome from small-scale expression evaluations in high-throughput mode is scalable to laboratory and industrial scales using well-established procedures. In this chapter, we offer a state-of-the-art perspective on currently available high-throughput pipelines for protein production in S. cerevisiae and P. pastoris and discuss future challenges and avenues for improvement.

Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins.

The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.

Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2.

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

Functional Analysis of Membrane Proteins Produced by Cell-Free Translation.

Cell-free production is a valuable and alternative method for the synthesis of membrane proteins. This system offers openness allowing the researchers to modify the reaction conditions without any boundaries. Additionally, the cell-free reactions are scalable from 20μL up to several mL, faster and suitable for the high-throughput protein production. Here, we present two cell-free systems derived from Escherichia coli (E. coli) and Spodoptera frugiperda (Sf21) lysates. In the case of the E. coli cell-free system, nanodiscs are used for the solubilization and purification of membrane proteins. In the case of the Sf21 system, endogenous microsomes with an active translocon complex are present within the lysates which facilitate the incorporation of the bacterial potassium channel KcsA within the microsomal membranes. Following cell-free synthesis, these microsomes are directly used for the functional analysis of membrane proteins.

Efficient production of antibody Fab fragment by transient gene expression in insect cells

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B.mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins.

High-Throughput Production of Proteins in E. coli for Structural Studies.

We have developed a standardized and efficient workflow for high-throughput (HT) protein expression in E. coli and parallel purification which can be tailored to the downstream application of the target proteins. It includes a one-step purification for the purposes of functional assays and a two-step protocol for crystallographic studies, with the option of on-column tag removal.

Autophosphorylation Assays Using Plant Receptor Kinases Synthesized in Cell-Free Systems.

The wheat germ cell-free protein synthesis system has a significant advantage for high-throughput production of eukaryotic multidomain proteins in a folded state. In this chapter, we describe two kinds of methods for performing autophosphorylation assay of plant receptor kinases (PRKs) by using the wheat cell-free system. One is an in vitro kinase assay performed using biotin-streptavidin affinity purification technology, and the other is a luminescence-based high-throughput assay for autophosphorylation analysis. We anticipate that our cell-free-based methods might facilitate the characterization of plant PRKs.

Engineering the transposition-based baculovirus expression vector system for higher efficiency protein production from insect cells

One of the most common methods for producing recombinant baculovirus for insect cell protein production involves a transposition mediated system invented over 2 decades ago. This Tn7-mediated system, commercially sold as Bac-to-Bac, has proven highly useful for construction of high quality baculovirus, but suffers from a number of drawbacks which reduce the efficiency of the process and limit its utility for high throughput protein production processes. We describe here the creation of Bac-2-the-Future, a 2nd generation Tn7-based system for recombinant baculovirus production which uses optimized expression vectors, new E. coli strains, and enhanced protocols to dramatically enhance the efficiency of the baculovirus production process. The new system which we describe eliminates the need for additional screening of positive clones, improves the efficiency of transposition, and reduces the cost and time required for high throughput baculovirus production. The system is compatible with multiple cloning methodologies, and has been demonstrated to produce baculovirus with equal or better titer and protein productivity than the currently available systems.

A cell-free expression and purification process for rapid production of protein biologics.

Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

Optimization and automation of an end-to-end high throughput microscale transient protein production process.

High throughput protein production from transient transfection of mammalian cells is used in multiple facets of research and development studies. Commonly used formats for these high number expressions are 12-, 24- and 96-well plates at various volumes. However there are no published examples of a 96-deep well plate microscale (1,000 μL) suspension process for mammalian transient expression. For this reason, we aimed to determine the optimal operating conditions for a high producing, microscale HEK293 transient system. We evaluated the hydrodynamic flow and measured the oxygen transfer rate (OTR) and transient protein expression for 96-deep well plates of different well geometries filled at 600-1,000 μL working volumes and agitated at various speeds and orbital diameters. Ultimately, a round well-round bottom (RR) 96-deep well plate with a working volume of 1,000 µL agitated at 1,000 RPM and a 3 mm orbital diameter yielded the highest and most consistent total transient protein production. As plate cultures are subject to evaporation, water loss from different plate seals was measured to identify an optimal plate sealing method. Finally, to enable higher capacity protein production, both expression and purification processes were automated. Functionality of this end-to-end automation workflow was demonstrated with the generation of high levels of human IgG1 antibodies (≥360 µg/mL) with reproducible productivity, product quality and ≥78% purification recovery.