rDNA Technology: A Brief History

In 1952, Joshua Lederberg proposed the term “plasmid” for an extrachromosomal genetic material that had been recently observed (Fig. 1) (Lederberg, 1952). At the time, no one could predict that the plasmid would one day become the workhorse at the center of countless recombinant technologies across a diverse range of industries. We know now that plasmids are circular, double-stranded DNA molecules naturally present in the cytoplasm of bacteria and other prokaryotes that are capable of replicating independently from the host cell (Fothergill-Gilmore, 1993; del Solar et al., 1998). Plasmids encode the information needed for their own replication and can confer specific characteristics to the host organism such as antibiotic resistance (Fothergill-Gilmore, 1993). However, research at the time of their discovery was hampered by a lack of viable strategies to introduce these circular double-stranded DNA molecules into cells (Patil et al., 2022). This research problem would lead to a monumental breakthrough for rDNA technologies by driving collaboration between experts in plasmid technology and targeted DNA manipulation (Patil et al., 2022). This collaboration and the intersection of these critical techniques resulted in the construction of the first biologically functional recombinant plasmid in vitro (Cohen et al., 1973) (Fig. 1). Restriction endonucleases EcoRI and EcoRII were used to cut the double-stranded plasmid, leaving single-stranded 3′ and 5′ sticky ends that could be joined together by DNA ligase to generate a circular double-stranded DNA molecule and functional plasmid (Cohen et al., 1973). This key experiment combined separate E. coli plasmid species pSC101 and pSC102, which contain genes encoding for resistance to tetracycline and kanamycin, respectively. The resulting biologically functional plasmid conferred resistance to both antibiotics, which was used to identify positive transformants via antibiotic treatment (Cohen et al., 1973). This represents the first use of what is commonly known now as a selection marker gene: a critical characteristic of plasmids and other cloning vectors that allows for selection of positively transformed cells. This approach utilizing selective advantage to identify positive transformants has proven invaluable to rDNA technologies and is still widely used today.

The following year, in 1974, this innovative strategy was successfully used to achieve the replication and transcription of eukaryotic Xenopus laevis DNA in E. coli (Morrow et al., 1974). Detection of X. laevis rDNA in E. coli transformants from this experiment demonstrated that E. coli could be used to clone genes with eukaryotic species of origin (Morrow et al., 1974). The next logical step was to test whether this approach could be employed to not only transcribe a eukaryotic gene but also to express and produce human proteins. Shortly after, the human hormone somatostatin was successfully cloned in E. coli using a pBR322 plasmid vector, demonstrating that rDNA technologies could be used to produce functional human proteins (Itakura et al., 1977) (Fig. 1). Only a few years later, Genentech’s recombinant human insulin named Humulin gained US FDA approval and entered the US market (Goeddel et al., 1979). This landmark advancement in rDNA technology would serve as the inspiration for the development of countless biotechnologies, showcasing vector-driven transformation in microorganisms like E. coli as a robust strategy to achieve the expression of a human gene product. With 621 biologic drugs currently FDA approved in the United States, it is clear these initial advances in rDNA technologies have inspired decades of innovative scientific research (FDA, 2021).

Expression Vectors: Characteristics and Examples

Since a bacterial plasmid was first used to express a eukaryotic gene in E. coli, our understanding of plasmids and other cloning vectors has greatly advanced. The bacterial plasmid was the first vector identified and optimized for the purpose of expressing recombinant gene products. However, there are also components present in the genomes of bacteriophages, yeasts, and viruses that can be used to accomplish effective transfer, cloning, and expression of a transgene in a heterologous system (Vieira and Messing, 1982; Patil et al., 2022). Specialized vectors that are optimized for transgene expression in a desired host organism are referred to as expression vectors and often contain additional noncoding sequences such as promoters, enhancers, and exons from the parent sequence that improve the transcription or expression of the gene product (Patil et al., 2022). Although vectors vary in their source, size, structure, capacity, and replication efficiency, all of them must retain several key attributes. To facilitate the discussion of key vector principles and their impact on successful transformation and expression of a gene insert, we refer to the widely used and iterated-upon plasmid pBR322 as an example (Fig. 2). This was one of the first vectors constructed with the intent of generating and selecting for recombinant clones, with pBR322 and its derivatives such as the pUC plasmids being commercially available and still widely used in rDNA technologies today (Balbás et al., 1986).

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Fig. 2.

Recombinant protein expression vectors: Characteristics and examples in P450 research. (A) First-generation vector pBR322 contains an ampicillin resistance gene (AmpR) and a tetracycline resistance gene (TcR), both shown in green. These genes serve as selection markers to identify positive transformants upon antibiotic treatment. The pBR322 plasmid contains a ColE1 ori site, which is illustrated in yellow. This ori site is recognized by E. coli host machinery to enable replication of the plasmid in this model organism. The repressor of primer (rop) gene illustrated in red encodes the rom protein, which limits the copy number of this vector. (B) An optimized expression vector derived from pCWOri+ is used to construct target plasmid toward heterologous P450 expression in E. coli (von Wachenfeldt et al., 1997). This vector contains two tac promoter sequences that are under transcriptional control of the lac repressor protein, which is illustrated in red (Barnes et al., 1991). The lac repressor is encoded by the lacI^q gene shown in green, which suppresses transcription until an inducing agent such as isopropyl β-D-thiogalactopyranoside is added. The vector and recombinant P450 gene insert are digested with endonucleases NdeI and EcoRI, enabling insertion and ligation of the transgene downstream of the two inducible tac promoters. This pCWOri+ derived expression vector also possesses two ori sites shown in yellow, which include the previously discussed ColE1 ori as well as an F1 ori that provides a recognition site for M13 bacteriophages.

Notable sequences used to construct the pBR322 plasmid include the ampicillin resistance gene (amp^R), a promoter from the ColE1-related plasmid named pRSF214, and the tetracycline resistance gene (tet^R) from the pSC101 plasmid discussed earlier (Cohen et al., 1973; Bolivar et al., 1977a,b) (Fig. 2). As established, cloning and expression vectors must contain a selection marker gene that confers a selective advantage to vector-bearing host cells (Cohen et al., 1973; Patil et al., 2022). Selection markers commonly encode antibacterial resistance or metabolic advantages, providing a survival advantage to positive transformants so they can be selected for through antibiotic treatment or nutrient deprivation (Bolivar et al., 1977b; Fothergill-Gilmore, 1993; Patil et al., 2022) (Fig. 2). Strategies to select for recombinant clones are not only limited to those providing a survival advantage to positive transformants, with other successful selection approaches utilizing targeted DNA insertion to functionally disrupt a gene (Bolivar et al., 1977b). This can confer traits to positive transformants that allow for their negative selection, such as tetracycline sensitivity in tet^R disruption and the formation of colorless colonies when the gene encoding the alpha protein of beta-galactosidase is disrupted (Bolivar et al., 1977b; Vieira and Messing, 1982).

In addition to containing a selection marker gene, a suitable expression vector must also be able to replicate in the host organism of choice. In plasmids, phagemids, and bacteriophage-based vectors, this is ensured through the inclusion of an origin of replication (ori) site (Patil et al., 2022). The ori site is recognized by host replication machinery, serving as a point of assembly for the components involved in DNA replication (del Solar et al., 1998; Patil et al., 2022). Some ori sites are specific to a certain host bacteria, while others are able to be recognized by the replication machinery of multiple species (Patil et al., 2022). The ori site in the pBR322 plasmid is derived from the ColE1 plasmid native to E. coli, making it a suitable choice for cloning and expression in this host organism (Bolivar et al., 1977b) (Fig. 2). The inclusion of more than one ori site in a cloning vector can allow replication across multiple different host species, expanding the flexibility of vectors for use in other model systems. The ori sites within expression vectors are often optimized for more efficient recruitment of replication components, with these sequences having a dramatic effect on the copy number of the vector (Friehs K, 2004; Patil et al., 2022). Another determinant of pBR322’s copy number is encoded by the repressor of primer (rop) gene shown in red (Cesareni et al., 1982; Banner et al., 1987) (Fig. 2). The protein encoded by this gene regulates plasmid copy number by promoting interaction of inhibitory RNA components that prevent primer maturation to modulate plasmid replication (Banner et al., 1987; Helmer-Citterich et al., 1988; del Solar and Espinosa, 2000). This limits the copy number of pBR322, a disadvantage in cloning that would be addressed by future derivatives of this vector.

Expression vectors must also contain multiple cloning sites that serve as unique locations for restriction endonucleases to recognize, enabling the specific digestion of the plasmid at these sites to generate a linearized double-stranded DNA molecule. The transgene of interest is digested using the same restriction enzymes to enable gene insertion into the plasmid. The compatible ends generated by restriction digest are joined together by DNA ligase, creating a recombinant vector that can undergo replication using host machinery (Fothergill-Gilmore, 1993; Patil et al., 2022) (Fig. 2). The pBR322 plasmid has been optimized to include a variety of multiple cloning sites for gene insertion, with many of them falling within critical segments of the vector, including both antibiotic resistance genes, the amp^R promoter, and the origin of replication (Bolivar et al., 1977b) (Fig. 2). The functional disruption of the tet^R gene via DNA insert that was discussed earlier utilizes the unique multiple cloning sites present within this gene. This allows for identification of host cells bearing the plasmid that contains the transgene DNA insert, with positive pBR322 transformants that successfully inserted and ligated the DNA fragment via HindIII, SalI, or BamHI endonucleases no longer having a functional tet^R gene (Bolivar et al., 1977b; Patil et al., 2022). This creative utilization of multiple cloning sites to generate specialized selection markers highlights the vast utility of restriction endonucleases, not only proving invaluable to the targeted insertion of a transgene but also in the customization of expression vectors and their features. While there are many context-specific applications of these enzymes and the cloning sites they recognize, this general strategy of enzyme-mediated transgene insertion, ligation, and replication has been widely applied in the cloning and expression of countless gene products as therapeutics and for functional study, including those integral to ADME processes such as P450 enzymes (Oeda et al., 1985; Unger et al., 1986; Gonzalez et al., 1988b) (Fig. 2).

In addition to the discussed components, an effective expression vector must also promote the efficient expression of the inserted transgene in the host system. Inclusion of a strong promoter that is compatible with the host species is critical to achieve adequate expression of a recombinant gene product (Fothergill-Gilmore, 1993). The promoter sequence must also be compatible with the modality of the gene product, with different promoters being used depending on whether the recombinant gene product is a protein, messenger RNA (mRNA), or small interfering RNA (siRNA; Patil et al., 2022). Some specialized expression vectors can also be designed with dual-promoter systems that aim to further increase the strength of transgene expression (Öztürk et al., 2017). These dual-promoter systems can consist of two twin promoters activated under the same conditions, as well as two nonidentical promoters activated during separate steps to aid production processes in biotechnology (Öztürk et al., 2017). Similarly, the inclusion of a strong termination sequence following the promoter and transgene can enhance expression and represents an important component of an efficient vector (West and Proudfoot, 2009; Gasanov et al., 2015; Patil et al., 2022). As recombinant technologies have continued to advance, expression vectors have become more readily adaptable tools that can be customized to efficiently express a wide variety of transgene products across many host systems. Recently developed expression vectors may also aid the purification of recombinant proteins through the installation of a polyhistidine affinity tag. Once purified, the affinity tag can be cleaved by tobacco etch virus protease to return the recombinant protein to its native conformation (Rocco et al., 2008). This strategy has been used to generate several novel expression vectors from the pET series capable of expressing and more easily purifying recombinant proteins (Rocco et al., 2008).

Like the choice of expression vector, choosing an appropriate host organism for transgene expression is a critically important step when utilizing rDNA technologies. Different model organisms possess distinct characteristics that can make them best suited to a particular application or gene product (Fothergill-Gilmore, 1993). Model organisms for recombinant expression can vary in their yield capacity, post-synthetic modification capability (glycosylation, acetylation), ability to secrete active proteins, and ability to express specific gene products or modalities. Common model species for transgene expression include bacteria (E. coli, Lactococcus lactis, Pseudomonas), yeast (Saccharomyces cerevisiae), mammalian cells (CHO, human embryonic kidney, COS), and insect cells (Sf9) (Koga et al., 1985; Oeda et al., 1985; Wurm, 2004; Chen, 2012). Research efforts continue to improve upon these expression systems and remedy their shortcomings. One of the most relevant examples of this is in E. coli, where advancements have enabled glycosylation of protein products and the use of antibiotic-free selection plasmids (Chen, 2012).

Applications of rDNA Technology in DMPK Research

Since the inception of rDNA technology, innovative researchers have been developing creative ways to adapt the rDNA platform to address their research questions with cDNA-expressed P450s (Fig. 1). One critical example of this was the development of site-specific mutagenesis methods utilizing M13 bacteriophage cloning vectors (Hutchison et al., 1978; Zoller and Smith, 1982). This method is used to elicit specific and targeted changes to a gene of interest for transformation into E. coli and other model organisms (Hutchison et al., 1978; Zoller and Smith, 1982). With the ability to introduce targeted single-nucleotide changes to any DNA fragment, researchers were able generate novel mutants for functional study. Often this approach is used to investigate the role of individual amino acid residues in contributing to the function or interactions of a specific protein. In DMPK research, this method has been used to generate novel P450 mutants to elucidate the role that specific residues play in protein behavior and enzyme function (Wilderman et al., 2012). Site-directed mutagenesis has also been widely applied in the characterization of other ADME related proteins such as ABC transporters and multidrug efflux pumps (Yao et al., 2013; Cox et al., 2018). Distinct from site-directed mutagenesis, random mutagenesis has also been used in DMPK research to generate novel CYP enzymes capable of withstanding harsh environmental conditions and performing biotransformations previously unseen in nature (Giver et al., 1998; Joo et al., 1999; Brandenberg et al., 2017; Arnold, 2018). Through many cycles of random mutagenesis and screening, beneficial mutations are accumulated that alter metabolic function and often promote an intrinsic promiscuity or side-reactivity of the natural enzyme (Arnold, 2018). This strategy developed by Frances Arnold is termed directed evolution, and it was awarded the 2018 Nobel Prize in chemistry. This innovative approach has been used to develop novel CYP enzymes capable of catalyzing many new and nonnatural reactions such as those valuable to medicinal chemistry or biotechnology processes (Brandenberg et al., 2017; Arnold, 2018).

Another valuable application of rDNA technologies in DMPK research has been improving the field’s access to robust research tools. A prominent example of this is the production of recombinant antibodies and nanobodies utilizing phage or yeast display libraries (Marks et al., 1991; McMahon et al., 2018). Specific and sensitive antibodies are critical reagents for any biomedical research, serving as important probes to detect and quantify target antigens. Recombinant antibodies may also serve as inhibitors to block specific protein-protein interactions, proving useful in elucidating the role of specific protein epitopes and interactions in determining protein function (Basu et al., 2019).

Previous methods of antibody production relied on hybridoma technology, requiring mature B cells be harvested from an animal immunized with the antigen (Basu et al., 2019). Because this relies on host immunization, the antibody generated is in response to host presentation of the antigen via the major histocompatibility complex. The antigen must undergo proteolysis to a smaller peptide for host presentation to occur, and because of this the antibodies generated by hybridoma methodology do not always successfully recognize the native conformation of the antigen they target (Roche and Furuta, 2015; Basu et al., 2019). However, when generating recombinant antibodies by phage display, the antigen of interest is present in its native conformation and is less likely to undergo alteration compared with hybridoma approaches (Basu et al., 2019). This represents a distinct advantage of recombinant antibody production when compared with previous hybridoma methods, especially when considering the importance of conformation in ADME-related protein function. Additionally, recombinant antibody production is significantly cheaper, does not require the use of animals, and allows for the inclusion of additional features such as an amino acid tag to aid the purification process (Basu et al., 2019).

This success utilizing rDNA technology to develop superior research tools has not only occurred with antibodies but has also extended to other modalities such as microRNAs (miRNAs), siRNAs, and aptamers (Ho and Yu, 2016). miRNAs are understood as epigenetic regulators with a vast array of targets and functions, with one miRNA able to regulate several mRNA transcripts at the posttranscriptional level (Ali Syeda et al., 2020; Yu et al., 2020). Understanding miRNA’s role in ADME gene regulation and function is critical to addressing a comprehensive picture of DMPK. The field of miRNA research has been somewhat limited by the widespread usage of chemically synthesized miRNA mimics as research tools. These commercially available miRNA mimics carry extensive chemical modifications that are needed to improve their stability and PK properties (Khvorova and Watts, 2017; Yu et al., 2019). The way in which these synthetic mimics are modified is not always disclosed, and modifications between manufacturers can vary in identity, location, and number (Yu et al., 2019). Although the nucleotide sequence is unaffected, these inconsistent modification practices generate miRNAs that are constitutively different from natural miRNAs and may lead to conflicting results between investigators (Yu et al., 2019; Diener et al., 2022; Traber and Yu, 2023).

To address these shortcomings, the Yu laboratory has developed an innovative bioengineering platform to express and purify recombinant miRNA molecules in E. coli (Li et al., 2021; Tu et al., 2021). Echoing the success of biologic proteins, these miRNAs are synthesized and folded inside living cells and better recapitulate the structures, modifications, and properties of endogenous miRNAs and thus represent superior tools for DMPK research and as potential therapeutics (Yu et al., 2019). Later in this review, we discuss the optimization of this recombinant strategy as well as its utilization in elucidating the impact of endogenous miRNA on ADME gene regulation and DMPK.

In addition to developing improved tools and reagents for research, advancements in rDNA technologies have also enabled novel approaches to generate improved cellular and animal models for the study of drug metabolism and human disease (Gonzalez and Yu, 2006; Dorr et al., 2017; Sundhari et al., 2019; Lu et al., 2021). One prominent advancement is the 2020 Nobel Prize winner in chemistry, an adaptable gene editing platform that utilizes clustered regularly interspaced short palindromic repeats (CRISPR) associated with a Cas9 endonuclease (Westermann et al., 2021). This promising technology commonly known as CRISPR-Cas9 has gained widespread popularity over the past decade, with many ex vivo gene therapies that use the platform currently undergoing clinical trials across a variety disease states (Li et al., 2020). In the context of DMPK research, this targeted gene editing technology has been used to generate valuable in vivo and in vitro knockouts to characterize individual ADME gene and allele function (Religia et al., 2021). Examples of ADME gene products knocked out or modified for functional study using CRISPR-Cas9 include CYP enzymes, ABC transporters, nuclear receptors, and solute carrier proteins (Karlgren et al., 2018; Wei et al., 2018; Liang et al., 2019; Karakus et al., 2020; Salanga et al., 2020; Lu et al., 2021). This approach can accomplish specific knockout of individual or multiple alleles of a target gene, allowing researchers to interrogate their role in xenobiotic metabolism, drug-drug interactions (DDIs), and PK (Dorr et al., 2017; Lu et al., 2021). While the CRISPR/Cas9 platform holds great promise to enable research and treat genetic disorders, future iterations of the technology must continue to improve upon its accuracy and precision as well as address concerns surrounding its unintended epigenetic impact (Doudna, 2020).

Microbial P450_CAM: A Window Into P450 Structure and Function

Despite some success with liver microsomes, the DMPK field was in need of a more suitable model system for the structural and functional characterization of individual P450 enzymes (Poulos et al., 1982). This role would be filled by the microbial cytochrome P450_CAM, a camphor-metabolizing monooxygenase that was identified in Pseudomonas putida (Gunsalus and Wagner, 1978; Poulos et al., 1982, 1987) (Table 1). This enzyme was purified for study prior to the advent of rDNA technologies and instead was isolated from bacterial culture according to a procedure optimized by Gunsalus and Wagner (Gunsalus and Wagner, 1978). P450_CAM was the first CYP enzyme that had been purified in both soluble and crystalline forms, making it an ideal biologic model to study P450 structure and function (Gunsalus and Wagner, 1978; Poulos et al., 1982, 1985). High-resolution crystal structures were generated of P450_CAM both bound to camphor and in the absence of its substrate, providing valuable insight into the substrate binding site and the structure-function relationship of the enzyme (Poulos et al., 1986, 1987). This crystallography data also elucidated the secondary structures and overall conformation of the polypeptide, illustrating important structural features of the enzyme such as the oxygen/heme binding pocket as well as a metal cation interaction that may stabilize active site formation (Poulos et al., 1987).

These advancements in P450_CAM visualization were also paired with amino acid sequencing techniques, further improving the detail of structural and functional characterization and enabling the comparison of P450_CAM to eukaryotic P450 sequences (Poulos et al., 1987). Compared P450 sequences were found to be fairly homologous, with residues that form the oxygen binding area being highly conserved between species (Poulos et al., 1987). Amino acid sequencing also revealed that eukaryotic P450s uniquely possess a hydrophobic NH2 terminus, a domain that was speculated at the time to contribute to the membrane solubility observed in mammalian P450s (Poulos et al., 1987). While bacterial cytochrome enzymes like P450_CAM are easily solubilized in the cytoplasm, eukaryotic P450 enzymes reside in the membranes of the endoplasmic reticulum and to a lesser extent the mitochondria (Lee et al., 1981). The functional role that this hydrophobic NH2 terminus plays in mammalian P450 membrane association and its effect on catalytic activity would later be elucidated using rDNA technologies (Oeda et al., 1985; Sakaguchi et al., 1987). P450_CAM has also served as an effective model system to assess the role of accessory proteins, with crystal structures of the enzyme complexed to its redox partner putidaredoxin providing insight into the structural changes induced by putidaredoxin and how they may facilitate electron transfer (Hiruma et al., 2013; Tripathi et al., 2013). Even today, P450_CAM is used as a model system to further investigate the allosteric roles of accessory proteins and how they may impact conformational dynamics (Poulos and Follmer, 2022).

Although P450_CAM was purified from a gram-negative bacterium, its extensive study aided our understanding of mammalian P450 enzymes by enabling the thorough characterization of well-conserved domains and functions (White et al., 1984; Black and Coon, 1987; Poulos et al., 1987; Gonzalez et al., 1988a,b; Umeno et al., 1988; Gonzalez and Gelboin, 1992). With isolation of pure P450 enzymes from human tissue being an arduous and time-consuming process, this P450_CAM model system was invaluable in establishing initial structure-function relationships and elucidating the basic catalytic mechanism conserved across nearly all P450s (Guengerich, 2020; Poulos and Follmer, 2022). P450_CAM was also one of the first P450 enzymes cloned for recombinant expression, utilizing a plasmid-derived vector under strict promoter control to produce catalytically active P450 monooxygenase in P. putida and E. coli host systems (Koga et al., 1985; Unger et al., 1986). This was not the only microbial P450 enzyme investigated utilizing rDNA technologies, as the crystal structure of the prokaryotic form of 14α-demethylase (CYP51) present in Mycobacterium tuberculosis was also elucidated this way (Podust et al., 2001) (Table 1). While CYP51 exists in most species as a membrane-bound enzyme that is localized to microsomes, the Mycobacterium tuberculosis genome possesses a uniquely soluble form of the enzyme that was expressed in E. coli for easier determination of the crystal structure (Bellamine et al., 1999; Podust et al., 2001). This newfound structural information would inform rational drug design of improved antifungal agents that target CYP51.

cDNA Directed Expression of Mammalian P450 Proteins

Not long after the crystal structure and amino acid sequence of P450_CAM were elucidated, rDNA strategies gained widespread usage in the study of mammalian CYP enzyme structure and function in drug metabolism (Fig. 1) (Sakaguchi et al., 1984; White et al., 1984; Oeda et al., 1985; Gonzalez et al., 1988a,b; Umeno et al., 1988; Larson et al., 1991b; Khan et al., 2002; Wester et al., 2002; Yu et al., 2002; Scott et al., 2003; Williams et al., 2003, 2004; Schoch et al., 2004) (Table 1). Although some P450 amino acid sequences had been elucidated through methods discussed earlier, the development of cloning, nucleotide sequencing, and DNA manipulation technologies would equip researchers with powerful tools to generate libraries of P450 cDNA sequences (Poulos et al., 1987; Gonzalez and Gelboin, 1992; Poulos and Follmer, 2022). Complementary DNA sequences for human P450 enzymes were generated using mature mRNA from liver and lung tissue lysates (Gonzalez and Gelboin, 1992). Purified mRNA was then used to generate cDNA strands via reverse transcriptase, which then underwent amplification by polymerase chain reaction to generate double-stranded DNA fragments (Patil et al., 2022). These gene segments were then ready be digested with complementary endonucleases and ligated into a vector for transformation of the amplified cDNA into a suitable host organism (Unger et al., 1986; Gonzalez and Gelboin, 1992; Patil et al., 2022). Due to observed sequence homology between species, human P450 sequences could be identified from these libraries through the use of cDNA probes and antibodies against rodent forms of P450 (Gonzalez et al., 1988b; Gonzalez and Gelboin, 1992). Once cDNA sequences of interest were identified and cloned using this approach, researchers set out to optimize the expression of these recombinant gene products in model organisms so their catalytic activities, substrate specificities, and structure-function relationships could be characterized (White et al., 1984; Oeda et al., 1985; Gonzalez et al., 1988a,b; Gonzalez and Gelboin, 1992; Schoch et al., 2004; Guengerich, 2020).

One important example that used cDNA-directed expression to illuminate P450 structure was the investigation of rabbit microsomal P450 and its cotranslational insertion into rough microsomes from dog pancreas (Sakaguchi et al., 1984, 1987) (Table 1). This would serve as an effective model of mammalian P450 integration into membranes of the endoplasmic reticulum, allowing researchers to interrogate the role of key structural features responsible for initiating cotranslational insertion and for conferring membrane solubility. Initial research demonstrated that cotranslational insertion of P450 could not occur without the presence of a ribonucleoprotein complex referred to as the signal recognition particle (SRP) (Sakaguchi et al., 1984). However, further investigation was required to identify the way in which microsomal P450 recognizes the SRP to influence P450 function and membrane topology. Hybrid cDNAs were generated to express chimeric forms of the P450 protein for study, with each mutant possessing unique modifications impacting the length and identity of the N-terminal amino acid chain (Sakaguchi et al., 1987). Of the several chimeric P450 proteins generated, only those possessing an NH2 terminus at least 29 AAs in length were able to cotranslationally insert into the membrane (Sakaguchi et al., 1987). This demonstrated that the insertion signal that enables SRP-mediated cotranslation to the membrane was contained within this 29 AA segment of the enzyme’s NH2 terminus (Sakaguchi et al., 1987). This research broadened the understanding of the NH2 terminus present in eukaryotic P450 forms, demonstrating the impact that NH2 truncation has on their topogenic function and membrane solubility (Sakaguchi et al., 1987).

Interestingly, recombinant research efforts expressing rat P450_MC in Saccharomyces cerevisiae also generated a truncated form of the enzyme that lacked an extended NH2 terminus (Oeda et al., 1985). Spectral and catalytic analysis revealed almost identical properties between the truncated and nontruncated forms of the enzyme, demonstrating that this NH2 terminus is not required for P450 catalytic activity (Oeda et al., 1985) (Table 1). This knowledge would be widely applied to solubilize mammalian P450 through deletion of the NH2 terminus, allowing researchers to more easily purify and crystallize mammalian P450 enzymes for structural and functional characterization (Larson et al., 1991b; Hsu et al., 1993; Richardson et al., 1995; von Wachenfeldt et al., 1997; Williams et al., 2000; Podust et al., 2001; Wester et al., 2002, 2003, 2004; Scott et al., 2003; Williams et al., 2003, 2004; Schoch et al., 2004; Rowland et al., 2006; Hsu et al., 2018). This cDNA-directed approach would soon be used to express truncated rabbit CYP2E1 in E. coli using a pKKHC vector, an optimized derivative of the pBR322 plasmid discussed earlier (Larson et al., 1991a,b) (Table 1). These research efforts further supported the notion that the NH2 terminus is not necessary to retain P450 monooxygenase function, with recombinant truncated CYP2E1 possessing identical spectral and catalytic properties compared with full-length CYP2E1 isolated from rabbit liver microsomes (Larson et al., 1991a,b).

Similar success was also achieved in the recombinant expression of rabbit CYP2C3 as a soluble dimer in E. coli. The CYP2C3 gene insert with a modified NH2 terminus was digested with endonucleases NdeI and EcoRI and transformed into an optimized pCWOri+ vector that had been linearized with the same enzymes (von Wachenfeldt et al., 1997) (Fig. 2). This general procedure is highlighted in Fig. 2, serving as an example of heterologous P450 expression in E. coli. Additionally, the key components of this vector that allow for strong transgene expression and inducible control are summarized in Fig. 2. This pCWOri+ derived expression vector would also later prove useful in the recombinant study of CYP2C5 (von Wachenfeldt et al., 1997; Wester et al., 2002, 2003).

Not long afterward, two mammalian P450 enzyme crystal structures (CYP2B4, CYP2C5) were identified through recombinant expression in E. coli (Wester et al., 2002, 2003; Scott et al., 2003) (Table 1). Both enzymes were expressed in soluble form through truncation of the NH2 terminus, allowing for their crystallization and structural characterization. Crystallization in the presence and in the absence of substrate allowed investigators to examine conformational differences that can determine substrate recognition and catalytic function (Wester et al., 2003). These experiments were also one of the first to add a histidine tag to the C terminus of the recombinant protein to aid the purification process (von Wachenfeldt et al., 1997). This strategy would also be employed in the study of human CYP2C8, which was uniquely expressed in E. coli as stable homodimer complexed by two palmitic acid molecules (Schoch et al., 2004). These peripheral fatty acid binding sites can alter characteristics of the active site and have potential to modulate substrate binding as well as contribute to DDIs (Schoch et al., 2004) (Table 1). This observed dimerization of CYP2C8 in addition to its significantly larger active site cavity suggest a distinct metabolic function of this enzyme when compared with previously elucidated P450s. This makes sense when considering that the primary substrates of CYP2C8 are comparatively larger molecules such as taxol (Schoch et al., 2004). Further research crystallized CYP2C8 with a variety of different substrates to examine the role of protein flexibility and ligand-induced conformational changes in accomplishing substrate oxidation (Schoch et al., 2008).

The crystal structure of human CYP2C9 was also determined through heterologous expression of a NH2 truncated mutant, which revealed a similarly large active site capable of accommodating multiple warfarin molecules or an additional substrate of similar size (Williams et al., 2003) (Table 1). Given that CYP2C9 can be activated by some exogenous compounds including warfarin, it was hypothesized that the binding of some ligands may serve an allosteric function that promotes enzyme activity (Williams et al., 2003). Compared with CYP2C9, the crystal structure of recombinantly expressed human CYP3A4 revealed a slightly smaller active site as well as a characteristic cluster of phenylalanine residues above it, some of which are known to cooperate in enzyme selectivity and function (Khan et al., 2002; Williams et al., 2004). X-ray crystallography data were generated for CYP3A4 in the absence of ligand, in the presence of an inhibitor metyrapone, and with its substrate progesterone (Williams et al., 2004) (Table 1).

To determine how the properties and structures of CYP3A4 and CYP3A5 differ, both enzymes were recombinantly expressed in E. coli and complexed with a high-affinity substrate ritonavir (Hsu et al., 2018). Crystallography data for these enzymes revealed differences in the shape and plasticity of their active sites upon substrate binding (Hsu et al., 2018) (Table 1). This structural data may reflect notable differences in the substrate dynamics and catalytic accessibility of these enzymes, potentially leading to divergent metabolic pathways or varying enzymatic efficiencies for shared metabolic pathways (Hsu et al., 2018). Additionally, enzymes of the CYP3A family can be differentially expressed across ethnic populations, with allelic variations having a significant effect on enzyme abundance and metabolic capability (Hirota et al., 2004; Yamaori et al., 2005). Given that the CYP3A family is the largest single contributor to drug metabolism, illuminating the structural features and processes that govern their catalytic activity and specificity is critically important in designing safe therapies, predicting DDIs, and understanding individual differences in drug metabolism (Zanger et al., 2008).

One recent advancement in the structural understanding of this enzyme family is the solved crystal structure of neonatal CYP3A7, a critical enzyme in hormone homeostasis and pediatric drug metabolism (Li and Lampe, 2019; Sevrioukova, 2021). Structural characterization of CYP3A7 was previously lacking, as the enzyme proved difficult to crystallize in its wild-type form. Using site-directed mutagenesis, a recombinant CYP3A7 variant capable of crystallization was generated to allow for structural characterization and comparison with other CYP3A enzymes (Sevrioukova, 2021) (Table 1). In many ways rDNA technologies have provided unprecedented insight into the catalytic structure, conformational dynamics, and substrate specificities of the most integral drug-metabolizing enzymes. Even today we continue to build upon our understanding of P450 monooxygenases, with recent investigations providing additional context regarding the nature of active site plasticity and redox partner selectivity (Poulos and Follmer, 2022).

Another cytochrome P450 enzyme that performs a significant share of xenobiotic biotransformations is human CYP2D6, as it is estimated to metabolize up to 20% to 30% of commonly prescribed drugs. The crystal structure was solved once again through heterogenous expression of a soluble recombinant CYP2D6 in E. coli utilizing a pCWOri+ derived vector (Rowland et al., 2006) (Table 1). This experiment revealed a structure most closely resembling that of CYP2C9, and the crystallography data enabled further research efforts toward building a more detailed understanding of the structural basis of ligand binding and specificity (Rowland et al., 2006). This information proved especially critical in understanding the many biotransformations mediated by CYP2D6 and CYP2C9, as their allelic variants between ethnic groups and polymorphisms between individuals can have a dramatic effect on metabolic activity, drug disposition, and therapeutic outcomes (Higashi et al., 2002; Yu et al., 2002; Ingelman-Sundberg, 2005; Zhou, 2009a,b). The rDNA platform has been adapted to interrogate the impact of specific CYP polymorphisms present in the population, uncovering mechanistic rationale for unique phenotypes of drug metabolism. The ability to recombinantly express specific P450 polymorphisms has allowed researchers to quantify enzymatic differences in the presence of any substrate, providing critical knowledge in our understanding of pharmacogenomic variability between individuals and populations. This knowledge has proven invaluable in drug design and clinical practice, as CYP polymorphisms and genetic variants can result in altered ADME processes, DDIs, and unexpected adverse effects. One key example of this is observed in warfarin-treated individuals with CYP2C9 polymorphisms, who are at increased risk of major bleeding events and excessive anticoagulation associated with increased drug exposure (Higashi et al., 2002). CYP2C9 polymorphisms have also been shown to promote genotype-dependent DDIs when warfarin or other major substrates are coadministered with an inducer or inhibitor of CYP2C9 (Higashi et al., 2002; Gardin et al., 2019; Cheng et al., 2022). Research utilizing rDNA technologies has revealed many genotype-specific differences in PK function and individual drug disposition, providing understanding that is critical to rational drug design, clinical dose management, and DDI prediction. Elucidating these pharmacogenomic differences has been key in developing and maintaining safe therapies, and this knowledge will prove increasingly useful as pharmacogenomic testing of patients becomes more commonplace and cost effective (Goh et al., 2017; Sayer et al., 2021).

In addition to providing valuable insight into the most predominant drug metabolizing P450s, rDNA technologies have also been used to characterize P450 enzymes that biosynthesize critical endogenous compounds. One predominant example of this is human CYP17A1, which is known to catalyze the synthesis of androgens and other important steroids (Miller and Auchus, 2011). Because of this, CYP17A1 inhibition represents an attractive strategy to prevent androgen synthesis and treat castration-resistant prostate cancer. The crystal structures of CYP17A1 bound to two inhibitors were determined by recombinant expression of a truncated in version in E. coli (DeVore and Scott, 2012) (Table 1). These initial findings elucidated the structural features integral to CYP17A1’s catalytic activity, further enhanced soon afterward by crystal structures of CYP17A1 with all four of its major endogenous substrates and clinically relevant inhibitors (Petrunak et al., 2014, 2017). Structural characterization of CYP17A1 enabled by recombinant expression provided critical information in the rational drug design of CYP17A1 inhibitors. Additionally, rDNA technologies allowed investigators to develop more selective CYP17A1 inhibitors through assessment of off-target interactions with other steroidogenic P450 enzymes such as CYP21A2 (Fehl et al., 2018).

Another enzyme that mediates critical biosynthetic reactions is cytochrome P450 aromatase (CYP19A1), which is known to synthesize estrogens from androgens. While the crystal structure of CYP19A1 was first determined using aromatase purified from human placenta, subsequent research efforts recombinantly expressed truncated CYP19A1 to further its structural characterization and interrogate the functional roles of specific amino acid residues (Ghosh et al., 2009, 2010; Lo et al., 2013) (Table 1). This insight into the structure of CYP19A1 and its androgen-specific active site aided the intelligent design of aromatase inhibitor drugs used to treat hormone receptor-positive breast cancers (Ghosh et al., 2012). Indeed, this same approach has proven similarly useful in the characterization of many P450 enzymes that catalyze the biosynthesis of important endogenous compounds (Brixius-Anderko and Scott, 2019, 2021; Liu et al., 2022). These investigations have supported the rational design of selective inhibitor drugs, with recent examples being the characterizations of P450 11B1, P450 11B2, and P450 8B1, which catalyze the production of cortisol, aldosterone, and bile acid, respectively (Brixius-Anderko and Scott, 2019, 2021; Liu et al., 2022) (Table 1).