PBTech_3_notes.pdf

# Bacterial gene expression and cloning This topic explores the design, implementation, and challenges of expressing proteins in bacterial systems, focusing on gene constructs, cloning, expression, and host selection. ## 1. Bacterial gene expression and cloning ### 1.1 Introduction to bacterial gene expression Bacterial gene expression refers to the process by which information from a gene is used in the synthesis of a functional gene product, often a protein. This is a fundamental process in molecular biology and biotechnology, enabling the production of recombinant proteins for research, therapeutic, and industrial purposes [2](#page=2). ### 1.2 Designing constructs for bacterial gene expression Designing an effective construct for bacterial gene expression involves selecting appropriate regulatory elements and the gene of interest (GoI) [27](#page=27). #### 1.2.1 Key components of expression plasmids Expression plasmids, such as those in the 'classic' pET system, contain several critical components: * **Origin of replication (ori):** Essential for plasmid replication and maintenance within the host cell [24](#page=24). * **Selection marker:** Typically an antibiotic resistance gene, used to select for cells that have taken up the plasmid [24](#page=24). * **Promoter:** Controls the initiation of transcription of the GoI. Promoters like T7 or lacUV5 are commonly used in inducible systems [27](#page=27) [7](#page=7). * **Multiple Cloning Site (MCS):** A region containing recognition sites for various restriction enzymes, facilitating the insertion of the GoI [6](#page=6). * **Ribosome Binding Site (RBS):** A sequence upstream of the start codon that facilitates ribosome binding for translation initiation [43](#page=43). * **Terminator:** Signals the termination of transcription [7](#page=7). * **Affinity tags:** Pre-cloned sequences (e.g., His-tags, GST) that facilitate protein purification [6](#page=6). #### 1.2.2 Inducible expression systems Inducible expression systems allow for controlled protein production, typically by using a promoter that is activated by a specific inducer molecule. The pET system, a widely used example, employs the T7 promoter, which is highly active but requires T7 RNA polymerase for transcription [7](#page=7). * **T7 RNA polymerase:** This polymerase is not naturally found in E. coli and is often introduced into the host strain via a lysogenic phage, such as DE3 (e.g., BL21(DE3)). The gene encoding T7 RNA polymerase is typically under the control of an inducible promoter, such as the lacUV5 promoter, which is regulated by IPTG (Isopropyl β-D-1-thiogalactopyranoside) [24](#page=24) [27](#page=27) [28](#page=28). * **IPTG induction:** IPTG is a structural homologue of allolactose and serves to de-repress the lac operon. It has a dual function: it can induce the expression of T7 RNA polymerase and it binds to the LacI repressor, thereby preventing it from binding to the lacO operator sequence, which is often located near the T7 promoter on the expression plasmid [27](#page=27) [28](#page=28). #### 1.2.3 Regulation and metabolic burden The expression of recombinant proteins can impose a significant "metabolic burden" on the bacterial cell, as transcription and translation are energy-intensive processes. This burden arises from the consumption of cellular resources such as DNA, RNA polymerases, ribosomes, and energy. If the burden is too high, it can lead to [39](#page=39) [40](#page=40) [41](#page=41): * Slower cell growth and replication rates [23](#page=23). * Plasmid loss, as cells without the plasmid gain a growth advantage [23](#page=23) [35](#page=35). * Accumulation of metabolic intermediates or redox imbalances [41](#page=41). * Even "escape mutants" where mutations reduce protein production to alleviate the burden [44](#page=44). To mitigate this, strategies like using weaker promoters or Ribosome Binding Sites (RBS), or employing auto-inducing media can be beneficial, balancing construct output with cellular capacity [39](#page=39) [43](#page=43). ### 1.3 Cloning and expression of proteins in bacterial cells The process of cloning and expressing proteins in bacteria involves several key steps: #### 1.3.1 Plasmid construction 1. **Ligation:** The GoI is inserted into a linearized expression vector, often using restriction enzymes and DNA ligase. The vector typically contains an origin of replication and a selection marker [6](#page=6). 2. **Transformation:** The recombinant plasmid is introduced into competent bacterial cells (e.g., E. coli) using methods like heat shock or electroporation [24](#page=24). 3. **Selection:** Transformed cells are plated on agar containing the appropriate antibiotic to select for cells that have successfully taken up the plasmid. #### 1.3.2 Protein expression 1. **Culture growth:** Selected colonies are grown in liquid culture to a suitable optical density. 2. **Induction:** The inducer (e.g., IPTG) is added to the culture to initiate the expression of the GoI, provided the expression system is inducible [26](#page=26) [27](#page=27). 3. **Incubation:** The culture is incubated for a period to allow for protein synthesis. 4. **Cell harvesting:** Cells are collected, usually by centrifugation [32](#page=32). #### 1.3.3 Protein purification 1. **Cell lysis:** The bacterial cells are disrupted to release the intracellular proteins. This can be achieved through physical methods (sonication, French press) or chemical methods (detergents, enzymes) [32](#page=32). 2. **Clarification:** Cell debris is removed, often by centrifugation. 3. **Purification:** The target protein is isolated from other cellular proteins. Affinity tags, which are often fused to the GoI, are crucial for this step, allowing for specific binding to a chromatography resin. Common methods include affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography [32](#page=32) [6](#page=6). #### 1.3.4 Protein localization Proteins can be expressed in different cellular compartments: * **Cytoplasm:** The most traditional location for expression [32](#page=32). * **Periplasm:** The space between the inner and outer membranes of Gram-negative bacteria. Proteins can be directed here using secretion signals [32](#page=32). * **Secretion:** Proteins can be exported out of the cell [32](#page=32). * **Membrane-anchored:** Proteins can be integrated into cellular membranes [32](#page=32). The compartment of expression can be genetically altered using specific protein tags that direct transport across membranes [32](#page=32). ### 1.4 Common expression difficulties and troubleshooting Troubleshooting protein expression involves a systematic approach to identify and resolve issues [37](#page=37). #### 1.4.1 Step 1: Sanity check * **Reagent integrity:** Ensure reagents are not expired or degraded. * **Dilution errors:** Verify correct preparation of media and inducer solutions. * **Plasmid/Host issues:** Check for mutations in the plasmid or the expression strain itself. * **Regulation control:** Confirm appropriate controls are used to assess expression (e.g., negative control without inducer). #### 1.4.2 Step 2: Protein not detected or not seen * **Low expression levels:** The protein might be produced at very low levels and thus be difficult to detect on a gel. This can be influenced by promoter strength, RBS efficiency, and codon usage. Additives to enhance expression or codon optimization might be necessary [38](#page=38) [53](#page=53). * **Complex mixture:** The target protein might be "hidden" among other proteins of similar size, especially if the expression is not very high. Loading optimization for SDS-PAGE is crucial [51](#page=51). * **Incorrect size:** The protein's apparent molecular weight on a gel might differ from the expected value due to amino acid composition biases, affecting migration [51](#page=51). #### 1.4.3 Step 3: Difficult to spot problems * **Rapid degradation:** The expressed protein may be unstable and quickly degraded by cellular proteases. Strategies include changing media, strains, localization signals, or using affinity tags [37](#page=37). * **Toxicity to host:** High-level expression of some proteins can be toxic to the bacterial cell, leading to a collapse in cell density upon induction, DNA errors, or colony morphology changes. Solutions include tighter expression regulation, compartmentalization, or zymogen expression [37](#page=37). * **Slow toxicity:** Protein levels might appear normal initially but decrease with repeated expression cycles. Fresh transformations and tight regulation are key [37](#page=37). * **Insoluble proteins:** Proteins may misfold and aggregate into inclusion bodies, making them difficult to extract and purify in their soluble form. Fusion partners (e.g., MBP, GST) that are highly soluble can sometimes improve the solubility of the protein of interest [54](#page=54). #### 1.4.4 Codon usage Different organisms have preferences for specific codons to translate amino acids. If a GoI contains codons that are rare in the expression host (e.g., E. coli), it can lead to slow translation and reduced protein expression. Optimizing the codon usage of the GoI to match the host's preferred codons can significantly enhance expression levels [53](#page=53) [54](#page=54). ### 1.5 Expression host platforms The choice of expression host significantly impacts protein expression, solubility, and post-translational modifications [78](#page=78). #### 1.5.1 E. coli * **Advantages:** Well-characterized genetics, rapid growth, inexpensive media, and a vast array of molecular tools and expression vectors (e.g., pET system). It is a traditional and widely used platform for cytoplasmic expression [22](#page=22) [23](#page=23) [32](#page=32). * **Disadvantages:** Lacks eukaryotic post-translational modification machinery like glycosylation, can struggle with folding of complex proteins, and may result in inclusion body formation [78](#page=78). #### 1.5.2 Other host platforms While E. coli is common, other hosts are used depending on the protein's requirements: * **Yeast (e.g., *S. cerevisiae*, *Pichia pastoris*):** Capable of some eukaryotic post-translational modifications, such as glycosylation. * **Insect cells (e.g., using baculovirus expression vectors):** Offer more complex eukaryotic post-translational modifications and are suitable for larger and more complex proteins. * **Mammalian cells:** Provide the most complete range of eukaryotic post-translational modifications, essential for many therapeutic proteins, but are more complex and expensive to culture. The selection of a host is influenced by factors such as biological constraints (e.g., requirement for specific modifications), technical feasibility (availability of molecular tools), application needs (yield, purity), regulatory aspects, and development costs [78](#page=78). #### 1.5.3 Considerations for host choice Key considerations when selecting an expression host include: * **Upstream factors:** Plasmid or genome integration, regulation mechanisms, expression levels, cell-to-cell variation, metabolic impact, and scalability [33](#page=33) [50](#page=50) [77](#page=77). * **Downstream factors:** Compartment of expression (intracellular, periplasmic, secretion), protein solubility, stability, purification methods, scale, and the need for affinity tags or additional sequences [33](#page=33) [50](#page=50) [77](#page=77). * **Constraining factors:** Biological requirements (e.g., glycosylation), technical development of molecular tools, application goals (yield, purity, contaminant impact), regulatory compliance (biocontainment), and development costs [78](#page=78). --- # Protein isolation and analysis techniques This section details the essential methodologies employed for isolating, purifying, and analyzing proteins, ranging from initial nucleic acid extraction to advanced biochemical assessments. ### 2.1 Nucleic acid extraction and manipulation for protein research The initial stages of protein isolation can involve working with nucleic acids, particularly RNA, to understand and potentially clone the gene of interest. #### 2.1.1 RNA extraction * **Purpose:** RNA extraction is a critical first step when the gene sequence of a target protein is unknown. RNA serves as the template for subsequent reverse transcription [10](#page=10). * **Methods:** Common methods involve chaotropic salts like 6 M Guanidinium chloride or acidified phenol:chloroform mixtures (e.g., TRIzol) [10](#page=10). * **mRNA Isolation:** While total RNA is extracted, messenger RNA (mRNA) is the primary target as it directly codes for proteins. mRNA constitutes only about 1% of cellular RNA, making its enrichment beneficial for downstream applications. Techniques like polyT capture, which leverages the natural polyA tail of eukaryotic mRNA, can be used to isolate mRNA [10](#page=10) [18](#page=18). #### 2.1.2 Reverse transcription (RT) * **Purpose:** Reverse transcription converts RNA into complementary DNA (cDNA). This process is typically a 1:1 conversion, meaning there is no inherent amplification of the nucleic acid [10](#page=10) [11](#page=11). * **Enzyme:** Reverse transcriptase (RT) is the enzyme used. Some RTs, like MMLV, possess terminal transferase activity and can template hop [17](#page=17) [18](#page=18). * **Primers:** A poly-dT primer is commonly used to bind to the polyA tail of mRNA for the synthesis of the first cDNA strand [12](#page=12) [18](#page=18). * **Establishing Known Ends for PCR:** To facilitate subsequent Polymerase Chain Reaction (PCR) and make cloning/sequencing more accessible, it's crucial to establish known DNA ends [11](#page=11). * **Poly-dT primer with overhang:** A poly-T primer can be designed with a specific overhang sequence that acts as a known upstream sequence. This overhang can contain recognition sites for restriction enzymes, such as BsaI, for downstream cloning strategies like Golden Gate assembly [17](#page=17) [20](#page=20) [21](#page=21). * **DNA/RNA hybrid oligo (SMART primer):** This primer is designed to facilitate template hopping of the reverse transcriptase. When the RT reaches the 5' end of the mRNA, it can "hop" onto the provided DNA/RNA hybrid oligo, extending it and creating a second known DNA end. This oligo is often made of DNA due to cost-effectiveness compared to RNA [17](#page=17) [18](#page=18) [20](#page=20). * **Double-stranded cDNA synthesis:** After the initial cDNA synthesis, cellular polymerases can synthesize the second strand. Alternatively, specific enzymatic treatments can be employed [13](#page=13). #### 2.1.3 Polymerase Chain Reaction (PCR) * **Purpose:** PCR is used to amplify the cDNA synthesized during reverse transcription. This amplification is essential because the amount of material after a single RT is often too small for efficient cloning or sequencing [11](#page=11) [21](#page=21). * **Requirements:** PCR requires two primers (forward and reverse) to enable logarithmic amplification. The known ends established during RT are used to design these primers [17](#page=17) [18](#page=18) [20](#page=20). * **Outcome:** Successful PCR yields enough double-stranded DNA material for subsequent cloning or sequencing [21](#page=21). #### 2.1.4 Cloning and Sequencing * **Cloning:** The amplified cDNA can be cloned into a suitable vector for further manipulation, storage, or expression. The original insulin cloning example utilized polyG and polyC overhangs on the plasmid and cDNA, respectively, to facilitate ligation [12](#page=12) [14](#page=14) [16](#page=16). * **Sequencing:** DNA sequencing is performed to determine the exact sequence of the isolated gene. Cloning first can be a "safer" approach for sequencing, especially when dealing with limited material or potential PCR failures, as DNA amounts are less likely to be limiting after cloning [10](#page=10) [12](#page=12). ### 2.2 Protein isolation and purification Once a protein of interest has been identified or engineered, it needs to be isolated and purified from the cellular milieu. #### 2.2.1 Cell lysis and protein solubilization * **Cell Isolation:** Cells are typically isolated using centrifugation [32](#page=32). * **Cell Disruption:** To release intracellular proteins, cells are disrupted using physical methods (e.g., sonication, French press) or chemical methods (e.g., detergents, enzymes) [32](#page=32). * **Solubility Considerations:** Proteins can be soluble or insoluble within the cell. Insoluble proteins may aggregate into inclusion bodies, often due to improper folding in a heterologous expression host lacking necessary chaperones. Detergents like SDS are crucial for denaturing and resuspending these aggregates for analysis [94](#page=94). #### 2.2.2 Electrophoretic separation techniques Electrophoresis is a cornerstone technique for separating proteins based on their physical properties. ##### 2.2.2.1 Agarose gel electrophoresis * **Matrix:** Agarose forms a large-pored matrix suitable for separating large molecules like DNA [87](#page=87). * **Principle:** An electric field drives DNA migration, primarily based on length due to its consistent charge-to-mass ratio [87](#page=87). * **Visualization:** DNA is visualized using intercalating dyes [87](#page=87). ##### 2.2.2.2 Polyacrylamide gel electrophoresis (PAGE) * **Matrix:** Polyacrylamide forms a covalently cross-linked matrix with smaller pores than agarose, allowing for finer separation [88](#page=88). * **Applications:** PAGE can be used for both nucleic acid and protein separation [88](#page=88). ##### 2.2.2.3 SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) * **Principle:** SDS-PAGE is specifically designed to separate proteins based primarily on their molecular weight [89](#page=89). * **Denaturation:** Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by disrupting non-covalent bonds, unfolding them into linear polypeptide chains [89](#page=89). * **Charge Introduction:** SDS coats the protein backbone, imparting a negative charge approximately proportional to its length, with about one negative charge per three amino acids. This creates a relatively constant charge-to-mass ratio across different proteins [89](#page=89). * **Electrophoretic Migration:** In the electric field, proteins migrate towards the anode (positive electrode). Due to the constant charge-to-mass ratio, smaller proteins encounter less resistance and migrate faster than larger proteins, allowing for size-based separation [89](#page=89) [90](#page=90) [91](#page=91). * **Gel Composition:** * **Acrylamide and Bisacrylamide:** A mixture of acrylamide and bisacrylamide is used to form the gel matrix. A ratio of 19:1 (acrylamide:bisacrylamide) is typical for protein separation [89](#page=89). * **Cross-linking:** Ammonium persulfate (APS) and TEMED are used to catalyze the free radical polymerization and cross-linking of acrylamide monomers [89](#page=89). * **Reducing Agents:** Reducing agents like DTT, β-mercaptoethanol, or TCEP are often included to break disulfide bonds, ensuring complete linearization of proteins [89](#page=89). * **Discontinuous Gel System:** * **Top (Stacking) Gel:** A low-density (4-6%) gel with a lower pH. Its primary function is to stack proteins into narrow bands at the interface with the lower gel, thereby improving resolution. Glycine in the running buffer does not function as a good electrolyte at this pH [90](#page=90) [91](#page=91) [92](#page=92). * **Lower (Resolving) Gel:** A higher density gel at a pH where both electrolytes (e.g., Tris and glycine) are charged and function efficiently. This is where the actual separation by mass/charge occurs [90](#page=90) [92](#page=92). * **Troubleshooting with SDS-PAGE:** SDS-PAGE is used to assess protein expression, confirm the expected molecular weight, and can help identify issues like degradation or toxicity. However, limitations exist, such as overlapping mobilities with endogenous proteins, low detection limits, and deviations in migration for small proteins or glycosylated proteins [51](#page=51) [95](#page=95) [99](#page=99). * **Visualization:** After electrophoresis, gels are stained with dyes like Coomassie Blue or SYPRO Orange to visualize protein bands [93](#page=93). ##### 2.2.2.4 Western blotting * **Purpose:** Western blotting is a technique used to detect specific proteins in a complex mixture, confirming their presence and sequence [96](#page=96). * **Procedure:** 1. **SDS-PAGE:** Proteins are first separated by SDS-PAGE [96](#page=96). 2. **Transfer:** Proteins are transferred from the PAGE gel to a solid support membrane, typically nitrocellulose or PVDF. This immobilization is crucial because proteins are denatured and can diffuse from the gel over time [96](#page=96) [97](#page=97). 3. **Blocking:** The membrane is blocked with an inert protein solution (e.g., non-fat dry milk or BSA) to prevent non-specific binding of antibodies [97](#page=97). 4. **Antibody Detection:** Specific antibodies are used to bind to the target protein. A primary antibody recognizes the protein of interest, and a secondary antibody, often conjugated to an enzyme (e.g., HRP) or a fluorescent tag, binds to the primary antibody [97](#page=97) [98](#page=98). 5. **Detection:** A substrate is added that reacts with the enzyme conjugate to produce a detectable signal (e.g., colorimetric, chemiluminescent), allowing visualization of the specific protein band on the membrane [98](#page=98). * **Comparison to ELISA:** While both use antibody-based detection, Western blotting involves prior separation of proteins by electrophoresis, allowing for analysis of molecular weight and post-translational modifications, which ELISA does not [100](#page=100). * **Limitations:** Western blotting confirms the presence of a linear epitope and the approximate molecular weight but does not directly indicate if the protein is correctly folded or functional [99](#page=99). ### 2.3 Biochemical and physicochemical analyses Beyond separation techniques, various analytical methods are employed to characterize proteins. * **Physicochemical and biochemical analyses** are crucial for assessing protein purity, determining the active fraction, comparing different batches (lot-to-lot variation), and evaluating long-term stability during production, storage, and transport . * **Comparison to endogenous proteins** can be a valuable approach for validation . * **Functional assays** can supplement Western blots to assess protein activity and folding status [99](#page=99). > **Tip:** Troubleshooting protein expression and isolation is an iterative process. Always start with basic checks (reagents, mutations, controls) before moving to more complex analyses [51](#page=51). > **Example:** When isolating insulin, early methods involved extracting RNA from insulinomas, followed by RT-PCR and cloning using methods like creating polyG and polyC overhangs on plasmids and cDNA to facilitate ligation. Modern approaches would leverage similar principles but with potentially more efficient and specific reagents [12](#page=12) [13](#page=13) [14](#page=14) [15](#page=15) [16](#page=16) [17](#page=17). --- # Metabolic burden and expression optimization Metabolic burden refers to the cost imposed on a bacterial cell when it is forced to express foreign proteins, impacting cellular capacity and yield, and necessitates strategies for optimization, including managing cell-to-cell variation and tuning gene expression [39](#page=39) [4](#page=4). ### 3.1 Understanding metabolic burden Metabolic burden arises from the cellular processes required for heterologous protein expression, which consumes essential resources like DNA, RNA polymerases, ribosomes, and energy from a common cellular pool. This forces the cell to expend energy on producing a protein that offers no evolutionary advantage, leading to a decrease in its overall fitness and growth rate [39](#page=39) [43](#page=43). #### 3.1.1 The cost of heterologous expression Heterologous expression involves compelling a cell to produce a non-native protein, often in large quantities. This is an energy-intensive process. The demands of transcription and translation, along with the maintenance of foreign DNA (e.g., plasmids), divert cellular resources [40](#page=40) [41](#page=41) [43](#page=43). * **Resource consumption:** Key cellular components such as DNA, RNA polymerases, ribosomes, and energy are finite and shared among all cellular processes [39](#page=39) [43](#page=43) [45](#page=45). * **Energy budget:** Cells operate within a maximum energy budget determined by their growth media. Introducing a foreign gene and inducing its expression significantly increases the demand on this budget [41](#page=41). * **Imbalances:** When a large fraction of the cellular budget is allocated to producing a foreign protein, insufficient resources remain for essential cellular functions, leading to imbalances. These can manifest as accumulating metabolic intermediates or redox imbalances due to slow replenishment of key enzymes. Even energy-dependent processes like nutrient import can be affected [41](#page=41). #### 3.1.2 Consequences of metabolic burden The increased metabolic load can lead to reduced cell growth rates and potential cellular stress responses. In extreme cases, the cell may struggle to cope, leading to the "rebellion" or even loss of the foreign DNA [40](#page=40) [41](#page=41). * **Reduced growth rate:** High demand for protein production can slow down cell replication [40](#page=40). * **Cellular stress:** The cell activates "alarm" responses to rebalance its metabolic budget [41](#page=41). * **Plasmid loss:** Prolonged or severe burden can lead to mutations and host/plasmid adaptations that reduce protein production per cell, freeing up resources for growth, or in more severe cases, lead to the loss of the plasmid altogether [44](#page=44). > **Tip:** Think of metabolic burden as asking a single person to perform multiple demanding tasks simultaneously; at some point, their performance will degrade, and they may become overwhelmed. > **Example:** A walker tasked with simply walking to a train station takes an hour. If asked to move their arms while walking, it might take slightly longer or be more tiring but manageable. However, if they are also asked to carry a heavy backpack, walk backward, and wear a costume, their ability to reach the station will be severely impacted, highlighting the concept of cumulative burden [40](#page=40). ### 3.2 Cellular capacity monitor A "cellular capacity monitor" is a concept from engineering applied to biological systems to gauge the metabolic burden on a cell. It is typically a low-cost reporter gene, such as GFP, expressed under a weak promoter and ribosome binding site (RBS) [39](#page=39) [40](#page=40) [45](#page=45). * **Function:** This monitor acts as an early warning system. If the cell begins committing significant capacity to a particular process (like expressing a protein of interest, PoI), the expression of the reporter gene will be readily affected, often dropping [40](#page=40). * **Purpose:** It helps researchers understand if the cell is under stress from high expression levels, serving as a "canary in a mine" [40](#page=40). ### 3.3 Optimizing expression and yield Optimizing foreign protein expression involves balancing the output of the desired protein with the cell's capacity to support it. This requires careful consideration of genetic elements and cellular resources. #### 3.3.1 Balancing construct output and cell capacity The goal is to achieve a simultaneous maximization of both construct output (the desired protein) and cell capacity (the cell's ability to function and grow). This is not a simple linear relationship; often, as cellular capacity is compromised to increase construct output, the overall yield can decrease [39](#page=39). * **Efficiency:** Defined as a measure of simultaneously maximizing construct output and cell capacity [39](#page=39). * **Optimal balance:** There exists an optimal point where a substantial portion of the cellular budget can be dedicated to protein production without completely sacrificing the cell's ability to perform its essential functions. For researchers, this means finding a sweet spot where the construct output is high, but enough capacity remains for the cell to avoid severe stress and maintain functionality [39](#page=39). #### 3.3.2 Role of genetic elements in optimization The design of expression constructs plays a crucial role in managing metabolic burden. * **Weaker RBS:** Using a weaker RBS can be beneficial in preventing overburdening the cell, particularly when combined with a strong promoter on a high-copy-number plasmid. A strong promoter generates many transcripts, and a strong RBS would lead to most ribosomes being occupied with translating the foreign gene. This monopolization of ribosomes can lead to imbalances and slow down the cell. A weaker RBS can moderate the rate of translation, allowing ribosomes to also support essential cellular functions [43](#page=43). * **Leaky expression:** Some expression systems can exhibit "leakiness," where a small amount of protein is expressed even in the absence of an inducer. This can be a factor to consider when designing strains or troubleshooting expression issues [31](#page=31). > **Tip:** When designing a construct, consider that stronger promoters and RBSs don't always equate to higher overall functional yields due to the potential for overwhelming the cell's metabolic capacity. > **Example:** In a system with a high copy number plasmid, a strong promoter, and a strong RBS, a cell might dedicate almost all its ribosomes to translating the foreign gene. This leads to a lack of essential proteins (metabolic enzymes, membrane components) and can result in imbalances, slowing growth and even triggering plasmid loss. Using a weaker RBS in this scenario can lead to higher yields of the foreign protein with better cell growth [43](#page=43). #### 3.3.3 Induced vs. uninduced constructs Distinguishing between induced and uninduced constructs is vital for understanding the costs associated with expression [41](#page=41). * **Uninduced construct:** Represents the baseline cost of harboring the foreign DNA, including plasmid replication, regulatory mechanisms, and any basal or "leaky" expression [41](#page=41). * **Induced construct:** Includes the costs of the uninduced construct plus the significant resource expenditure required for active protein synthesis. Induction typically involves adding a specific chemical inducer (e.g., IPTG or arabinose) to activate gene expression from a regulated promoter [40](#page=40) [41](#page=41). ### 3.4 Cell-to-cell variation and expression tuning Gene expression in bacterial populations is not a uniform, continuous process but rather a stochastic and variable one, exhibiting cell-to-cell and temporal variations [46](#page=46). #### 3.4.1 Noise in gene expression "Noise" in gene expression refers to the inherent randomness in biological processes, especially when dealing with small numbers of molecules [46](#page=46). * **Stochasticity:** Events like transcription factor binding or the initiation of transcription involve discrete steps with associated probabilities. This means that even under the same conditions, individual cells can have different expression levels due to random fluctuations [46](#page=46). * **Analogy:** It's like flipping a coin; while an unbiased coin has a 50/50 chance of heads or tails, you cannot predict the exact number of throws needed to get a head, nor can you guarantee an equal number of heads and tails in a small series of throws [46](#page=46). #### 3.4.2 Tuning gene expression Tuning refers to the correlation between the amount of inducer present and the resulting level of gene expression. This can be observed at both the individual cell level and the population level [46](#page=46). * **Population tuning:** Involves increasing the overall amount of product produced across the entire population of cells, or by increasing the fraction of cells that are producing the protein [46](#page=46). * **Cell tuning:** Represents a scenario where some cells might become high producers while others remain non-producers, even with the same inducer concentration. This can have significant implications for metabolic burden, as non-expressing cells may outgrow strongly expressing ones [47](#page=47). > **Example:** Imagine a population of 10 cells. > * **Uninduced:** All 10 cells express zero reporter protein . > * **Scenario 1 (Cell tuning):** After induction, 5 cells express at a high level, and 5 remain at zero expression. The average expression is 1 . > * **Scenario 2 (Population tuning):** After induction, all 10 cells express at a moderate level. The average expression is also 1 . > While the population average is the same, the per-cell distribution of expression is very different, impacting the metabolic burden differently [47](#page=47). * **Tunable inducers:** Some inducers are described as 'tunable,' implying that their concentration can be adjusted to control the level of expression, offering a mechanism to manage metabolic burden [46](#page=46). --- # Expression hosts and host engineering This section explores the diverse range of expression hosts utilized for protein production and the advanced techniques of host engineering to enhance protein expression and functionality. ### 4.1 Overview of expression considerations When selecting an expression host, several factors influence the upstream and downstream processes of protein production [77](#page=77). * **Upstream considerations** include the nature of the plasmid or genome used, regulatory elements, expression levels, cell-to-cell variation, metabolic impact on the host, and the desired scale of production [77](#page=77). * **Downstream considerations** involve the protein's compartment (intracellular, periplasmic, or secretion), solubility, stability, the chosen purification method, scale, and the use of affinity tags or additional sequences [77](#page=77). These considerations are often constrained by biological, technical, application-specific, and regulatory factors. Biological constraints include essential post-translational modifications (PTMs) for proper protein folding. Technical constraints relate to the availability and development of molecular tools for a specific host. Application requirements focus on yield, purity, and the impact of potential contaminants. Regulatory aspects, such as biocontainment, and development costs are also critical [78](#page=78). ### 4.2 Complexity of expression hosts Expression hosts range in complexity from cell-free systems to single-celled organisms and multicellular organisms. The spectrum includes [80](#page=80): * **Cell-free systems:** These systems utilize cellular extracts to perform protein synthesis outside of living cells. * **Single-cell organisms:** * Bacteria (e.g., *Escherichia coli*, *Corynebacterium glutamicum*, *Lactobacillus* spp., *Pseudomonas putida*, *Vibrio natriegens*) [83](#page=83). * Yeasts (e.g., *Saccharomyces cerevisiae*, *Pichia pastoris*) [83](#page=83). * Algae [80](#page=80). * Archaea [80](#page=80). * **Multicellular organisms:** * Insect cells [80](#page=80). * Mammalian cells (e.g., Chinese hamster ovary (CHO), Human embryonic kidney (HEK)) (#page=80, 83) [80](#page=80) [83](#page=83). * Transgenic plants [80](#page=80). * Transgenic animals [80](#page=80). > **Tip:** *Escherichia coli* is known for issues with protease accumulation and acetate imbalance, which can lead to toxicity [83](#page=83). ### 4.3 Prokaryotic expression hosts Prokaryotes, such as *Escherichia coli*, offer significant advantages for large-scale protein expression: * **Strengths:** * Easy to manipulate genetically and develop [83](#page=83). * Capable of large-scale fermentation [83](#page=83). * Generally achieve high yields, with the desired protein sometimes constituting up to 20% of the culture's dry weight [83](#page=83). * Simple and inexpensive feedstock requirements [83](#page=83). * **Limitations:** * Exhibit minimal post-translational modifications (PTMs) [83](#page=83). * Provide the lowest support for proper protein folding [83](#page=83). * PTMs can differ significantly from human PTMs, which can be a limitation for therapeutic proteins where specific modifications are required for function or to minimize immunogenicity [83](#page=83). > **Example:** A "high yield" in bacterial and yeast systems means achieving grams per liter (g/L) scale in protein synthesis, which translates to a lower cost of production per mass of expressed protein [83](#page=83). ### 4.4 Yeast expression hosts Yeasts, including *Saccharomyces cerevisiae* and *Pichia pastoris*, are also widely used for protein expression: * **Strengths:** * Relatively easy to manipulate genetically [83](#page=83). * Can be grown in large-scale fermentations [83](#page=83). * Can achieve high yields [83](#page=83). * Are capable of performing PTMs, though these can differ from human PTMs [83](#page=83). * **Limitations:** * Can have slower growth rates compared to bacteria [83](#page=83). * Scaling up can be more challenging [83](#page=83). * Can be more expensive to culture [83](#page=83). ### 4.5 Mammalian cell expression hosts Mammalian cells, such as Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells, are crucial for producing complex therapeutic proteins that require human-like PTMs: * **Strengths:** * Support all necessary PTMs, which are critical for protein function and immunogenicity [83](#page=83). * Can be grown in suspension, facilitating scale-up [83](#page=83). * **Limitations:** * Can be slow to grow [83](#page=83). * Scaling up can be difficult [83](#page=83). * Often require expensive, serum-supplemented media [83](#page=83). ### 4.6 Transgenic organisms for protein production Transgenic plants and animals can be engineered to produce proteins, particularly for large-scale applications. #### 4.6.1 Therapeutic production in transgenic milk The host genome can be engineered to introduce a gene of interest (GoI), which is the gene encoding the desired protein. Promoters can be used to restrict expression to specific tissues, such as the mammary gland for milk production [81](#page=81). > **Example:** In therapeutic production using transgenic milk, the GoI would be the gene coding for a therapeutic protein. For instance, spider silk genes could be introduced into a goat to produce spider silk in its milk [81](#page=81). However, proteins can sometimes leak into the organism's circulation, potentially affecting animal health [81](#page=81). #### 4.6.2 Case study: Human antithrombin III (hAT) in goat's milk The production of human antithrombin III (hAT) in goat's milk is a notable example [82](#page=82). * **Company Evolution:** The companies involved in its development have undergone several name changes, illustrating the dynamic nature of the biopharmaceutical industry (e.g., Genezyme Transgenics Corporation  GTC Biotherapeutics  rEVO  LFB Biotechnologies) [82](#page=82). * **Product:** Human antithrombin III (hAT) is a 52 kDa serpin (serine protease inhibitor) [82](#page=82). * **Characteristics of rhAT in Goat's Milk:** * Exhibits a modified glycosylation pattern compared to native hAT [82](#page=82). * Has a 7 to 10-fold lower serum half-life [82](#page=82). * Possesses a 4-fold higher heparin affinity [82](#page=82). * **Limitations:** Not suitable for patients allergic to goat albumins [82](#page=82). ### 4.7 Host engineering Host engineering involves modifying the host organism to improve protein production and introduce desired functionalities. This can include altering the host's genetic code or incorporating enzymes from different hosts [84](#page=84). * **Deeper host engineering** refers to modifying the host to access functionalities not typically present, going beyond the introduction of the gene of interest [84](#page=84). * **Examples of host engineering:** * **Yeast glycosylation:** Engineering yeast strains to exhibit a "humanized" glycosylation pattern, which is much closer to the human one, can address limitations in producing proteins like IgG [84](#page=84). * **Incorporation of unnatural amino acids:** Modifying the organism's genetic code to allow the incorporation of chemically modified or unnatural amino acids [84](#page=84). * **Cytoplasmic environment modification:** Alterations, such as those seen in CHO cell engineering, can address issues like the reducing environment in the cytoplasm, which can be detrimental for certain protein productions [85](#page=85). > **Tip:** Host engineering aims to overcome perceived limitations of a particular host. By combining engineered traits, it is possible to develop hosts that are significantly better suited for producing specific recombinant proteins (#page=84, 85 [84](#page=84) [85](#page=85). ### 4.8 Impact of host engineering on biopharmaceutical production The landscape of biopharmaceutical production is vast, with numerous approved and developing biologics. Deeper host engineering has the potential to significantly alter the balance of preferred expression systems. By enabling modifications that were previously considered limitations, such as achieving human-like PTMs or optimizing cellular environments, host engineering can lead to more efficient and effective production of complex protein therapeutics (#page=84, 85 [84](#page=84) [85](#page=85). --- ## Common mistakes to avoid - Review all topics thoroughly before exams - Pay attention to formulas and key definitions - Practice with examples provided in each section - Don't memorize without understanding the underlying concepts

| Term | Definition | |------|------------| | Construct | A designed DNA molecule, often a plasmid, engineered for a specific purpose such as bacterial gene expression. It contains regulatory elements and the gene of interest for manipulation and expression within a host cell. | | Cloning | The process of isolating a specific gene or DNA fragment and inserting it into a vector, which is then replicated within a host organism to produce multiple copies of the DNA. This is fundamental for gene expression and study. | | Gene of Interest (GoI) | The specific gene that a researcher wishes to express or study within a bacterial system. This gene encodes the protein of interest. | | Protein of Interest (PoI) | The protein encoded by the gene of interest (GoI) that a researcher aims to produce through bacterial expression and subsequent purification. | | Plasmid | A small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells and are commonly used as vectors in molecular biology for gene cloning and expression. | | Multiple Cloning Sites (MCS) | A short region in a cloning vector that contains a sequence of DNA with several unique restriction enzyme recognition sites. This allows for the insertion of foreign DNA fragments into the vector at multiple positions. | | Affinity Tag | A short peptide sequence or protein fused to a protein of interest to facilitate its purification. These tags bind specifically to a particular ligand or matrix, enabling easy separation of the target protein from other cellular components. | | T7 RNA Polymerase | A highly efficient RNA polymerase from the bacteriophage T7. It is often used in bacterial expression systems to drive high levels of transcription from T7 promoters, leading to significant protein production. | | T7 Promoter | A DNA sequence recognized and bound by T7 RNA polymerase, initiating transcription. It is a strong promoter commonly used in expression vectors to achieve high levels of gene expression in bacteria. | | Lac Operon | A genetic unit in bacteria that encodes genes for the metabolism of lactose. It is a classic example of gene regulation, typically induced by lactose or its analog, IPTG. | | LacI (Lacl repressor) | A protein that acts as a repressor in the lac operon system. It binds to the operator region, blocking transcription unless an inducer molecule is present. | | LacO (Lac Operator) | A DNA sequence within the lac operon to which the LacI repressor protein binds. This binding prevents RNA polymerase from initiating transcription of the operon's genes. | | IPTG (Isopropyl β-D-1-thiogalactopyranoside) | A synthetic inducer molecule that mimics the natural inducer of the lac operon, allolactose. It binds to the LacI repressor, causing it to detach from the operator and allow transcription of genes under the control of the lac promoter. | | Induction | The process of activating gene expression, often by adding a specific inducer molecule (like IPTG) to the bacterial culture. This triggers the transcription and translation of the gene of interest. | | Expression Host | A specific strain of bacteria, such as E. coli, chosen for its ability to express foreign genes and produce proteins. Different host strains have various characteristics that can impact protein yield and quality. | | BL21(DE3) | A common E. coli expression strain that contains a copy of the T7 RNA polymerase gene integrated into its genome via a lambda DE3 lysogen. This strain is particularly well-suited for high-level protein expression using T7 promoter-based vectors. | | Plasmid Loss | The phenomenon where bacterial cells fail to retain a plasmid during growth. This can occur due to errors in replication and segregation or when cells without the plasmid have a growth advantage. | | Metabolic Burden | The physiological cost to a bacterial cell resulting from the expression of foreign genes or the maintenance of plasmids. This burden can divert cellular resources, impacting growth and overall fitness. | | Origin of Replication (ori) | A specific DNA sequence on a plasmid where DNA replication begins. It is essential for the plasmid to be replicated and maintained within the bacterial host cell. | | Selection Marker | A gene present on a plasmid that confers a selectable trait, such as antibiotic resistance. This allows for the identification and selection of bacterial cells that have successfully taken up the plasmid. | | Antibiotic Resistance Marker | A type of selection marker that confers resistance to a specific antibiotic. Bacteria containing the plasmid with this marker can survive and grow in the presence of the antibiotic, while those without it are killed. | | Cytoplasmic Expression | The production of a recombinant protein directly within the cytoplasm of a bacterial cell. This is a common method for protein expression, though the protein may need to be purified from the cell lysate. | | Periplasm | The compartment between the inner and outer membranes of Gram-negative bacteria. Proteins can be directed to the periplasm for expression and isolation, which can sometimes aid in folding or stability. | | Secretion | The process by which a protein is exported from the bacterial cell into the surrounding medium. This can facilitate purification and avoid issues related to intracellular accumulation or toxicity. | | Upstream Processing | All steps in bioprocessing that occur before the expression of the target protein. This includes gene cloning, construct design, and optimization of expression conditions. | | Downstream Processing | All steps in bioprocessing that occur after the expression of the target protein. This involves the isolation, purification, and formulation of the expressed protein. | | Codon Usage | The differential frequency of synonymous codons used to encode amino acids in different organisms. Optimizing codon usage can improve protein expression levels in a heterologous host. | | Ribosome Binding Site (RBS) | A sequence on messenger RNA (mRNA) that initiates the process of translation. The strength of the RBS influences the rate of translation and thus protein production. | | Leaky Expression | A low level of gene expression that occurs even in the absence of an inducer. This can be due to incomplete repression or spontaneous transcription. | | T7 Lysozyme | A protein co-expressed with T7 RNA polymerase in some systems, which can bind to and inhibit T7 RNA polymerase activity. It is used to reduce leaky expression by titrating out small amounts of T7 RNA polymerase. | | Cell-to-cell Variation | Differences in gene expression levels or cellular behavior observed among individual cells within a bacterial population, even under the same conditions. This is often due to stochastic events in gene regulation. | | Noise (in gene expression) | The random fluctuations in gene expression levels within a cell or population. This arises from the inherent probabilistic nature of molecular interactions in biological systems. | | Tuning | The process of adjusting the level of gene expression in response to specific stimuli or conditions. It can occur at the cell level or the population level. | | Logic Gates | Biological circuits designed to mimic the behavior of electronic logic gates, taking multiple inputs and producing an output based on predefined logical rules. These are used for complex gene regulation. | | Heterologous Expression | The expression of a gene or protein from one organism in a different host organism. For example, expressing a human gene in E. coli. | | Fusion Partner | A protein that is genetically fused to a protein of interest. Fusion partners can be used to improve solubility, facilitate purification, or aid in protein folding. | | Solubility | The ability of a protein to dissolve in a solvent, such as an aqueous buffer. In protein expression, achieving soluble protein is crucial for downstream applications. | | Codon Optimization | The process of altering the DNA sequence of a gene so that it uses codons that are frequently employed by the expression host organism. This can significantly enhance protein production. | | Ribosome Stalling | A temporary halt in the translation process by a ribosome, often caused by the presence of rare codons or issues with tRNA availability. This can sometimes be exploited to improve protein folding. | | RNA Extraction | The process of isolating RNA molecules from cells or tissues, serving as a template for subsequent molecular biology techniques. | | Reverse Transcription | A process where an enzyme, reverse transcriptase, synthesizes a complementary DNA (cDNA) strand from an RNA template. | | PCR (Polymerase Chain Reaction) | A laboratory technique used to amplify specific segments of DNA, creating millions of copies from a small initial amount. | | DNA/RNA Hybrid Oligo | A short, synthetic strand of nucleic acid composed of both DNA and RNA components, often used to facilitate specific molecular interactions or as a primer. | | Agarose Gel Electrophoresis | A technique used to separate DNA fragments based on their size and electrical charge by passing them through a gel matrix made of agarose. | | Polyacrylamide Gel Electrophoresis (PAGE) | A technique used to separate proteins or nucleic acids based on their size and electrical charge by passing them through a gel matrix made of polyacrylamide. | | SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) | A widely used technique for separating proteins based on their molecular weight by denaturing them with SDS and separating them through a polyacrylamide gel. | | Western Blotting | A technique used to detect specific proteins in a sample by transferring them from a gel to a membrane and then using antibodies to identify and bind to the target protein. | | Biochemical Analyses | The study and measurement of the chemical properties and reactions of biological molecules, often used to characterize purified proteins. | | Reverse Transcriptase (RT) | An enzyme that synthesizes a DNA molecule from an RNA template through the process of reverse transcription. | | SMART oligo | A proprietary hybrid RNA/DNA primer designed for template hopping during reverse transcription, enabling 5'-end sequence generation. | | Terminal Transferase (TdT) | An enzyme that adds nucleotides to the 3' end of a DNA strand without the need for a template, often used to create overhangs for cloning. | | Poly-dT primer | A short oligonucleotide sequence consisting of thymine bases, used to initiate reverse transcription by binding to the poly-A tail of mRNA. | | Poly-G overhang | A sequence of guanine nucleotides added to the 3' end of a DNA strand, often created by terminal transferase, to facilitate ligation into a cloning vector. | | Poly-C overhang | A sequence of cytosine nucleotides added to the 3' end of a DNA strand, often created by terminal transferase, to facilitate ligation into a cloning vector. | | Electrophoresis | A laboratory technique that uses an electric field to separate molecules based on their size and electrical charge as they migrate through a medium. | | Denaturation | The process of disrupting the three-dimensional structure of a protein or nucleic acid, often through chemical or thermal means, leading to loss of function. | | Anode | The positive electrode in an electrochemical cell, towards which negatively charged ions (anions) migrate. | | Cathode | The negative electrode in an electrochemical cell, towards which positively charged ions (cations) migrate. | | Stacking Gel | The upper, less dense gel layer in a discontinuous gel electrophoresis system, designed to concentrate and compress protein samples before they enter the resolving gel. | | Resolving Gel | The lower, more dense gel layer in a discontinuous gel electrophoresis system, where the actual separation of proteins by size occurs. | | Coomassie Blue | A common stain used to visualize proteins in polyacrylamide gels, binding to the proteins and making them visible as colored bands. | | SYPRO Orange | A fluorescent dye used to stain proteins in gels, offering higher sensitivity than Coomassie Blue for visualization. | | Inclusion Body | Aggregates of misfolded or insoluble proteins that form within bacterial cells during recombinant protein expression. | | Nitrocellulose Membrane | A type of membrane material commonly used in Western blotting to immobilize proteins transferred from a gel for subsequent antibody detection. | | Blocking Step | A procedure in blotting techniques (like Western blotting) where unoccupied sites on the membrane are coated with blocking agents to prevent non-specific binding of antibodies. | | Antibody | A protein produced by the immune system that specifically binds to a particular antigen, used in blotting techniques to detect specific proteins. | | Physicochemical Analyses | Analyses that study the physical and chemical properties of substances, often used to characterize purified proteins. | | Lot-to-lot Variation | Differences observed in the quality or characteristics of a product manufactured in different production batches. | | Cellular Capacity Monitor | A reporter gene, such as GFP, used to gauge the metabolic burden on a cell. Expressed with a weak promoter and ribosome binding site (RBS), it is designed to be sensitive to changes in cellular resources. A drop in reporter expression indicates that the cell is committing its capacity to another process, like the expression of a protein of interest, and is under stress. | | Induced Cultures | Bacterial cultures that have undergone the process of activation for the expression of a gene of interest (GoI). This activation is typically triggered by the addition of an inducer molecule, after which the cultures are referred to as induced. | | Uninduced Cultures | Bacterial cultures prior to the addition of an inducer molecule. In this state, the expression of the gene of interest is not yet activated, providing a baseline measurement of cellular costs associated with harboring a plasmid and potentially leaky expression. | | Tuning (Gene Expression) | A phenomenon that describes a correlation between the amount of an inducer present and the level of gene expression achieved. This can manifest at the population level by increasing protein production in every cell or by increasing the proportion of cells within the population that are actively producing the protein. | | Noise (Gene Expression) | The inherent stochasticity and variability observed in biological processes, particularly at the molecular level where discrete events with probabilities of success occur. This can lead to spontaneous gene expression even without external regulatory signals, analogous to unpredictable coin tosses. | | Cell Tuning | A scenario where, in a population of cells, a specific inducer level results in some cells becoming high producers of a protein while others remain non-producers. This variation in response at the individual cell level is distinct from population tuning where expression levels are more uniformly distributed. | | Population Tuning | A scenario where, in response to an inducer, a population of cells exhibits a consistent increase in protein expression across all or most cells. This differs from cell tuning, where the response is more heterogeneous, with some cells showing high production and others little to none. | | Expression Hosts | Organisms or cellular systems utilized for the production of recombinant proteins, chosen based on factors like protein folding, post-translational modifications, ease of manipulation, scale-up potential, yield, and cost. | | Host Engineering | The process of modifying the genetic makeup or cellular machinery of an expression host to enhance protein production, improve protein functionality, or introduce novel characteristics for specific applications. | | Post-Translational Modification (PTM) | Biochemical modifications that occur to a protein after its synthesis on the ribosome, which can include glycosylation, phosphorylation, and disulfide bond formation, crucial for protein folding, function, and stability. | | Yield | The amount of purified protein obtained relative to the mass of cells or the volume of culture used; a higher yield translates to a more cost-effective production process per unit of expressed protein. | | Biocontainment | Measures and strategies employed to prevent the unintended release or spread of genetically modified organisms (GMOs) or biological agents from a laboratory or production facility into the environment. | | Glycosylation | A type of post-translational modification where carbohydrate chains (glycans) are covalently attached to a protein, significantly influencing its folding, stability, solubility, and biological activity. | | Intracellular Expression | The production of a recombinant protein within the cytoplasm or nucleus of a host cell, requiring subsequent extraction and purification from the cell lysate. | | Periplasmic Expression | The production of a recombinant protein in the periplasm, a compartment located between the inner and outer membranes of Gram-negative bacteria, offering advantages in terms of protein folding and disulfide bond formation. | | Transgenic Organisms | Organisms that have been genetically modified by the introduction of DNA from another organism, allowing them to express foreign genes and produce specific proteins, such as therapeutic proteins in milk. | | Prokaryotes | Single-celled microorganisms that lack a membrane-bound nucleus and other organelles, commonly used as expression hosts due to their rapid growth, ease of manipulation, and high protein yields; examples include *Escherichia coli* and *Corynebacterium glutamicum*. | | Eukaryotes | Organisms whose cells contain a nucleus and other membrane-bound organelles, including yeast, insect cells, and mammalian cells, which offer more complex post-translational modification capabilities than prokaryotes. | | Yeast | A type of eukaryotic microorganism widely used as an expression host, known for its ability to perform post-translational modifications and grow in large-scale fermentation; examples include *Saccharomyces cerevisiae* and *Pichia pastoris*. | | Insect Cells | Eukaryotic cells derived from insects, often used for the expression of complex recombinant proteins that require specific post-translational modifications, particularly glycosylation, which can be achieved using baculovirus expression systems. | | Mammalian Cells | Eukaryotic cells derived from mammals, such as Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) cells, which are capable of performing human-like post-translational modifications, making them ideal for producing therapeutic proteins. | | Transgenic Plants | Plants that have been genetically modified to express foreign genes, allowing for the production of recombinant proteins in seeds, leaves, or other plant tissues, offering a scalable and potentially cost-effective production system. | | Transgenic Animals | Animals that have been genetically modified to express foreign genes, with potential applications for producing therapeutic proteins in their milk, blood, or eggs, though ethical and regulatory considerations are significant. | | Unnatural Amino Acids | Amino acids that are not among the standard 20 genetically encoded amino acids, which can be incorporated into proteins through host engineering to expand the functional diversity and chemical properties of recombinant proteins. |