High-Throughput Spectroscopic Analysis of mRNA Capping Level.

Eukaryotic mRNAs are characterized by terminal 5' cap structures and 3' polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a novel biosensor method designed to detect the presence of both cap structures and polyadenylation sites on mRNA molecules. This novel biosensor is sensitive to mRNA degradation and can quantitatively determine capping levels of mRNA molecules within a mixture of capped and uncapped mRNA molecules. The biosensor displays a constant dynamic range between 254 nt and 6507 nt with reproducible sensitivity to increases in capping level of at least 20% and a limit of detection of 2.4 pmol of mRNA. Overall, the biosensor can provide key information about mRNA quality before mammalian cell transfection.

Cell-Free Systems for the Production of Glycoproteins.

Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction.

Cell-Free Protein Synthesis Using Pichia pastoris.

Pichia pastoris (syn. Komagataella phaffii) is an industrially relevant recombinant protein platform that has been used to produce over 5000 proteins to date. Cell-free protein synthesis can be used as a screening tool before strain development or for the production of proteins that are difficult or toxic to make in vivo. Here we describe the methods for generating an active cell lysate from P. pastoris using high pressure homogenization and an improved reaction mix which results in high yields of reporter proteins such as luciferase, and complex proteins such as human serum albumin and virus-like particles.

Expression of proteins in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is currently one of the most versatile and popular hosts for the production of heterologous proteins, including industrial enzymes. The popularity of P. pastoris stems from its ability to grow to high cell densities, producing high titers of secreted heterologous protein with very low amounts of endogenous proteins. Its ability to express correctly folded proteins with post-translational modifications makes it an excellent candidate for the production of biopharmaceuticals. In addition, production in P. pastoris typically uses the strong, methanol-inducible and tightly regulated promoter (PAOX1), which can result in heterologous protein that constitutes up to 30% of total cell protein upon growth in methanol. In this chapter, we present methodology for the production of secreted recombinant proteins in P. pastoris, and we discuss alternatives to enhance protein production with the desired yield and quality.

Production and purification of an active CRM197 in Pichia pastoris and its immunological characterization using a Vi-typhoid antigen vaccine.

CRM197 is a commonly used glycoconjugate carrier that improves the immunogenicity of vaccines, particularly in infants. Despite the advantages of this diphtheria toxoid mutant, low yields, production in inclusion bodies, and the requirement for specific growth conditions have limited the breadth of successful recombinant protein expression platforms available for its expression. We evaluated Pichia pastoris as a production host, using the methanol inducible AOX1 promoter and a modified α-mating factor signal peptide for secretion into the supernatant. Final purified yields >100 mg L-1 culture were achieved when produced in a bioreactor, which is equivalent to the productivity obtained from bioprocesses using the native Corynebacterium diphtheriae host. Recombinant CRM197 was purified to ≥95% homogeneity and showed the expected endonuclease activity. Furthermore, mice immunized with a Salmonella enterica serovar Typhi capsular Vi antigen conjugated to our recombinant CRM197 showed greater than 5-fold increase in immune response. Overall, the results demonstrate that Pichia pastoris is a suitable expression host for the production of high quality CRM197 for vaccine applications.

Knockout of RSN1, TVP18 or CSC1-2 causes perturbation of Golgi cisternae in Pichia pastoris.

The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.

Methods for Expression of Recombinant Proteins Using a Pichia pastoris Cell-Free System.

Cell-free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell-free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell-free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post-translational modifications. Here, we describe methods for conducting cell-free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription-translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike. © 2020 The Authors. Basic Protocol 1: Preparation of Pichia pastoris cell lysate Basic Protocol 2: Coupled in vitro transcription and translation Basic Protocol 3: Determining luciferase production from cell-free protein synthesis reactions Alternate Protocol 1: Linked in vitro transcription and translation Alternate Protocol 2: Quantifying HSA protein concentration Support Protocol 1: Preparation of mRNA by in vitro transcription for linked transcription and translation.

Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort.

A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System.

Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform.

Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 µg ml -1 . Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling.

Adaptation allows organisms to maintain a constant internal environment, which is optimised for growth. The unfolded protein response (UPR) is an example of a feedback loop that maintains endoplasmic reticulum (ER) homeostasis, and is characteristic of how adaptation is often mediated by transcriptional networks. The more recent discovery of asymmetric division in maintaining ER homeostasis, however, is an example of how alternative non-transcriptional pathways can exist, but are overlooked by gold standard transcriptomic or proteomic population-based assays. In this study, we have used a combination of fluorescent reporters, flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER homeostasis. Under low ER stress, asymmetric division leaves daughter cells with an ER deficiency, necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before growth. Mathematical analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter cells. These results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.

Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria.

Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization.

Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L-1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.

Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris.

Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.

Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris.

The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 µg mL-1 in small scale expression. VRC01 was expressed using both the α-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly, using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the α-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.

Rapid screening of cellular stress responses in recombinant Pichia pastoris strains using metabolite profiling.

Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P. pastoris clones.

The Commonalities in Bacterial Effector Inhibition of Apoptosis.

Antiapoptotic pathways of the host cell play integral roles in bacterial pathogenesis, with inhibition of those pathways resulting in halted disease pathology. Certain pathogens have developed elegant mechanisms to modulate the fate of the host cell, many of which target novel pathways that are poorly understood in the context of the cell biology. Bacterial pathogenesis research not only promotes the understanding of the role of antiapoptotic pathways in bacterial infection, but has a broader context in understanding the epitome of human disease, that is, developing the understanding of tumorigenic or inflammatory pathways. Here we review host antiapoptotic signalling pathways manipulated by translocated bacterial effectors that propagate the disease state, drawing common parallels and showing the novel differences.

Liquid PTVA: a faster and cheaper alternative for generating multi-copy clones in Pichia pastoris.

BACKGROUND: Multiple cognate gene copy clones have often been used in order to increase the yield of recombinant protein expression in the yeast Pichia pastoris. The method of posttransformational vector amplification (PTVA) has allowed for the efficient generation of multi-copy clones in P. pastoris. However, despite its relative ease and success, this process can be expensive and time consuming. RESULTS: We have developed a modified version of PTVA, called Liquid PTVA, which allows for faster and cheaper selection of multi-copy clones. Cultures are grown in liquid medium with only a final selection carried out on agar plates, reducing overall antibiotic usage and increasing the speed of clone amplification. In addition, it was established that starting PTVA with a single copy clone resulted in higher copy number strains for both traditional plate PTVA and liquid PTVA. Furthermore, using the Zeocin selection marker in liquid PTVA results in strains with higher growth rates, which could be beneficial for recombinant protein production processes. CONCLUSIONS: We present a methodology for creating multi-copy clones that can be achieved over 12 days instead of the traditional 45 and at approximately half the cost.

Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.

RESULTS: We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. CONCLUSION: Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.

Can too many copies spoil the broth?

The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields that can be achieved due to high volumetric productivity. However, low specific productivity still inhibits the potential success of this platform. Multi-(gene) copy clones are potentially a quick and convenient method to increase recombinant protein titer, yet they are not without their pitfalls. It has been more than twenty years since the first reported use of multi-copy clones and it is still an active area of research to find the fastest and most efficient method for generating these strains. It has also become apparent that there is not always a linear correlation between copy number and protein titer, leading to in-depth investigations into how to minimize the negative impact of secretory stress and achieve clonal stability.