Changing culture conditions to tune expression
Before some of the cloning and engineering strategies previously discussed, “simpler” approaches may also prove helpful. High-yields and high-quality are not necessarily linked and higher-yields may, in fact, produce large quantities of unfolded, non-functional protein. When this outcome is not intended, for example, if avoiding inclusion body formation is desired, lowering the culture temperature during protein induction can lead to higher yields of functional, high-quality material (Francis & Page, 2010; Li et al., 2001; San-Miguel et al., 2013; Weickert et al., 1997). This has mostly been explored in E. coli, however, and little is reported on low temperature cultivation effects in yeast. The use of synthetic media also facilitates slowed expression and production compared to nutrient-rich media. Lastly, like strong promoters, growth conditions can also affect plasmid copy number (Stueber & Bujard, 1982).
A promising field that has gained much traction over the last decade is the use of CF systems for high-yield expression. Once used primarily as a research tool to understand transcription and translation has now been adapted to produce protein products at scale for synthetic biology (Garenne et al., 2021; Silverman et al., 2020). CF systems do not suffer from the production bottlenecks presented by their whole-cell counterparts, but there are other important considerations. The intracellular systems and processes that accompany traditional in vivo expression have effectively been removed, leaving only the essential protein expression and production machinery. Prokaryotic CF systems emerged using components isolated from E. coli where recombinantly expressed, purified, components have been used successfully – “Protein synthesis Using Recombinant Elements” (PURE system) (E. D. Carlson et al., 2012; Klammt et al., 2006; Ohashi et al., 2010). Success has also been met using crude cell lysis extract (Kigawa et al., 2004). Eukaryotic CF expression has been developed from a variety of sources ranging from wheat germ to Chinese hamster ovary (CHO) cells to HeLa cells (Anderson et al., 1989; Brödel et al., 2014; Harbers, 2014; Weber et al., 1975). There are only a few reports of CF systems using expression machinery isolated from yeast and most utilize soluble proteins to demonstrate feasibility of the system (Hodgman & Jewett, 2013). Extracts from E. coli have been utilized successfully in CF expression on a multi-liter scale (Figure 3). The reactions are stable for several hours and can be scaled to produce gram-level quantities of material (E. D. Carlson et al., 2012). CF yeast systems have also been utilized successfully to produce active firefly luciferase at µg-scale (Hodgman & Jewett, 2013). While most of the large-scale production efforts described utilize soluble proteins, strategies to adapt this technique to MPs are summarized nicely in several reviews (Klammt et al., 2006; Sachse et al., 2014). CF In Vitro Transcription/Translation reaction (IVTT) systems provide a solution to the potentially limited space available at the plasma membrane that is necessary to support properly folded, functional MPs. Arguably, it also leaves proteins less susceptible to the structural and conformational defects that arise from overcrowding in the membrane during overexpression. Rather, in the absence of the native cell-membrane, CF systems rely on artificially supplemented media to support MP solubility. Mild detergents and other lipids that assume a bilayered morphological structure are added to the synthetic milieu to keep newly synthesized hydrophobic MPs soluble and conformationally active (Klammt et al., 2006). CF systems streamline the production process by omitting the need for any additional protein refolding protocols to be established prior to characterization. One important consideration is the compatibility of detergent and lipid additives not only to the target protein, but also their effects, if any, on the viability of the IVTT reaction components. Also, the concentration of detergent and lipids required to maintain protein solubility and active reaction components must be determined as well. Detergents must be maintained at a concentration above their critical micelle concentration, CMC, to maintain effective solubility properties (Kalipatnapu & Chattopadhyay, 2005; Sachse et al., 2014). Though this is perhaps a slight simplification of the complex biophysical interactions between MPs and solubilizing detergents and lipids. At any given time during the course of the reaction, the concentration of freely available detergent and associated micelles fluctuates during expression as proteins are synthesized and occupy micelle aggregates. Further complicating this process are the changes in by-product accumulation accompanying the reaction. These are important considerations when optimizing the synthesis reaction, but advantageously, carrying this out in a CF system also provides flexibility in the amount of detergent that can be added, changing the solubilizing capacity of the reaction mixture. Generally, nonionic detergents such as Brij and Triton X-100 detergents can be scaled at minimal cost. Other proprietary detergent mixtures such as Empigen BB®, which is comprised of a heterogeneous distribution of varying chain length molecules, is also mild and nondenaturing (Lowthert et al., 1995). These types of detergents are least likely to cripple the functionality of the components in the reaction mixture leading to longer reaction times and increased protein yields. Further, orchestrating a system to remove reaction by-products as they accumulate and adding substrates can facilitate longer reaction times and improve yields (Schoborg et al., 2014). Detergent solubilized proteins can be later reconstituted into bilayered systems for downstream analysis (Rieth et al., 2020).
Conclusions and Future Directions
A plethora of strategies along with synthetic biology tools for high-yield recombinant MP production is described. Integral MPs remain one of the most challenging expression targets due to their extreme hydrophobicity, which presents challenges to most host organisms when expressed at high levels. Overexpression often involves recruiting rigorous gene expression conditions that lead to activation of stress-related intracellular pathways in yeast that can thwart efforts to achieve high yields. Engineering native and nonnative hybrid promoters can help alleviate these problems as well as coupling activator and repressor sites upstream of the target protein. Consideration of terminator sequences provides another way to fine-tune expression and slow the coupled transcription-translation process to help avoid overwhelming the intracellular translation machinery. Many of the strategies summarized in this regard relate to the use of expression plasmids to achieve maximum yields. Indirect approaches have also been described using metabolic engineering and other genome engineering techniques to generate KO and KI strains with the aim of redirecting yeast metabolism and nutrient resources toward producing the target protein. The use of metabolic/genome engineering to halt non-essential processes has been demonstrably successful in some cases for soluble proteins and has yet to be applied to MP expression. Generating KOs to remove unwanted protease activity can also help increase yields, although increasing yields is not always coupled with high-quality, functional proteins. This review is intended to summarize the tools available to MP researchers looking to produce proteins for characterization. It describes best approaches that can be applied to maximize yields, which is often a bottleneck to progress in areas of structural biology and other types of analyses. It is also intended to help define areas in need of further development and where future efforts could be beneficially directed so that more can be learned about the processes critical to expression. While still somewhat a new methodology, CF expression for MPs holds tremendous promise as an alternative to heterologous expression in microbes by removing some of the critical barriers to overexpression and maximizing yields. Further, similar strategies used for synthetic biology in microbial systems have been attempted in CF systems with some success. Genetic components; transcription/translation, promoters, regulatory elements are beginning to be explored in CF systems and already make use of well-characterized, robust systems such as the E. coli -based T7 transcription system (Garenne et al., 2021). Much of the progress seen with CF systems has been derived from E. coli . However, the same limitations present in E. coli expression still plagues its CF counterpart. Namely, the ability to carry out PTMs. Although, less is known about CF systems using yeast-derived components much of its progress has been pioneered by Jewett and co-workers and could potentially complement E. coli -derived systems for this reason, but without the limitations of the finite space in the plasma membrane. Along these lines, it may be advantageous to use CF in tandem with microbial systems by testing an expression system in vitro prior to transferring the genetic components to a cell-based system. CF systems are amenable to laboratory automation and potential scale-up making high-throughput testing possible, which can help supplement arduous efforts to screen single expression conditions at one time. CF also makes incorporation of unnatural amino acids possible for biophysical and other downstream analysis.