Lecture 8 - BMB 470

Protein Expression: From Gene to Protein

BMB 470 Lecture 8
October 27, 2025

Goals of the Upcoming Weeks

Overall focus on understanding how recombinant proteins are expressed in bacterial systems, purified, and analyzed for both functional and structural properties.
Overview of lectures related to recombinant DNA techniques, protein expression, purification, and characterization.
Today’s focus (Lecture 8) and preview of upcoming topics (Lectures 9-12).

Lecture 8 Outline

Why make and study proteins?
Using Bacteria as Protein Factories
- Why bacteria?
- Key features of bacterial expression systems
- Challenges and limitations
The pET Expression System
- Overview
- Key components
- How it works
- Preview of Experiment 8A&B

Importance of Studying Proteins

Proteins function as the "molecular machines" of the cell, driving virtually all cellular processes.
Understanding proteins equates to understanding biology as they are vital for various functions:
- Actin and myosin in muscles create movement.
- Hemoglobin carries oxygen in the blood to fuel body activities.
- Na+/K+ pump generates electrical signals for heartbeats.
- Opioid receptors in the brain modulate pain and mood.

Application and Relevance

Why Produce Proteins in the Lab?
- Initiating any understanding or usage of proteins requires their production.
- Key Areas of Focus:
  - Structure: What does it look like?
  - Function: What does it do?
  - Mechanism: How does it carry out its function?
Research and Industry Applications:
- Use of proteins in everyday life and therapeutics to prevent or treat diseases.

Native vs. Recombinant Protein Sources

Protein Source	Description	Pros	Cons
Native Protein	Purified directly from the organism where it naturally occurs.	- Naturally folded

Biologically active | - Low yield
Impure
Difficult to purify |
| Recombinant Protein | Produced by introducing the gene of interest into a model organism (e.g., bacteria, yeast, mammalian cells). | - Scalable
High yield
Consistent quality
Modifiable | - May require optimization for proper folding or activity |
Recombinant proteins are often preferred due to controlled, scalable production and ease of modification for research, industrial, or therapeutic applications.

Expression Systems Overview

The choice of expression system depends on the complexity, yield, and intended use of the protein of interest:
- Pros and Cons associated with different systems (E. coli, Yeast, Insect Cells, and Mammalian Cells).

Using E. coli as Protein Factories

E. coli is the most commonly used and best-characterized organism for protein expression, offering the following advantages:
- Fast growth and easy to culture, allowing for quick protein production.
- Low cost in terms of media and equipment, translating to high yield from small culture volumes.
- Simple genetics for easy manipulation of DNA for expression.

Molecular Mechanisms in E. coli

Native Protein Expression in E. coli

Transcription:
- Carried out by RNA polymerase starting at the promoter and stopping at the terminator sequence.
Translation:
- Initiated when the ribosome binds to the ribosome-binding site (RBS) and recognizes start codon (AUG), terminating at the stop codon (e.g. UAA).

Recombinant Protein Expression in E. coli

Expression Plasmid: Contains the gene of interest, with key features:
1. Carries the gene of interest.
2. Inducible promoter controlling protein production.
3. Affinity tags that facilitate protein purification.

Limitations of Bacterial Expression Systems

Challenges include:
- Lack of complex post-translational modifications (PTMs) (e.g., glycosylation, phosphorylation).
- Protein misfolding potential.
- Toxicity of some proteins to the host.
- Variance in codon usage between target gene and bacterial host.
Despite these limitations, it is possible to express eukaryotic proteins within E. coli through proper vector design linking the eukaryotic gene to bacterial regulatory sequences.

The pET Expression System

Known as Plasmid for Expression by T7 RNA polymerase
This system facilitates high levels of recombinant protein production in E. coli:
- The gene of interest is inserted into a pET vector which is then introduced into E. coli for expression.

Key Components of the pET System

pET Vector: Contains instructions for both transcription and translation.
Host Cell: Provides the necessary protein machinery for transcription and translation processes.

The pET Vectors

Key Features include:
- Origin of replication (ORI)
- Antibiotic resistance marker (AmpR)
- T7 promoter linked to the gene of interest, controlling transcription initiation.
- lac operator for transcription regulation via the lac repressor, which blocks access to the promoter unless IPTG is present.

Mechanism of Action in the pET System

T7 Promoter & RNA Polymerase:
- T7 RNA Polymerase provides high transcription rates and specificity towards T7 promoter.
- Distinct from E. coli RNA polymerase, the T7 RNA polymerase recognizes a fixed 17 bp region rather than -10/-35 promoter regions.
Transcription Regulation:
- For transcription to occur, T7 RNA polymerase must bind to the T7 promoter, which is regulated by the binding state of the lac repressor and presence of the inducer IPTG.

Regulation of Transcription in the pET System

IPTG, a non-degradable analog of allolactose, binds the lac repressor, causing it to dissociate from the lac operator and promote transcription of target genes by T7 RNA polymerase.

Inducible Protein Expression Process

Absence of IPTG (‘OFF’ State):
- Without IPTG, the lac repressor binds to lacO, blocking transcription.
Presence of IPTG (‘ON’ State):
- Presence of IPTG inactivates the lac repressor, allowing transcription to proceed.

Key Considerations for Protein Expression Timing

IPTG should be added when bacterial cultures reach mid-log phase growth (typically at OD600 = 0.6-0.8 in LB media). This timing ensures sufficient biomass for high protein production.

Advantages of the pET System

High yield of proteins due to efficient T7 RNA polymerase.
High specificity of T7 promoters guarantees focused transcription.
Controlled expression through IPTG, along with enhanced purification ease via incorporation of tags like His-tag for affinity chromatography.

Review of Key Ideas and Considerations

Engaging students through a quiz (Kahoot! Quiz Competition) to reinforce learnings from today.

Overview of Workflow Phases

Detailed timelines for mutagenesis, production, and analysis phases that will be executed over the upcoming weeks.
Week 1: Preparation and Mutagenesis 1.
Week 2: Production and Experiment 8A: Colony Screening
Week 3: Analysis and Experiment 8B: Protein Expression by IPTG Induction.

Important Reminders and Upcoming Assessments

Quiz #4 this Friday.
Ensure to test LockDown browser on D2L before quizzes.
One-page cheat sheet is allowed for the quiz.
Bring a calculator and ensure phones are away for exams.
Lab notebooks for experiments 7A-8B due next week.
Lab reports are due on November 21.

Component	Role & Interplay	Present (Normal Function)	Absent	Mutated
pET Vector	Carries the gene of interest (GOI) and regulatory elements crucial for high-level, inducible expression.	Plasmid present, allowing for gene expression.	No recombinant protein expression; host cells remain wild-type.	Depending on the mutation, expression may be impaired or altered.
- Origin of Replication (ORI)	Specific DNA sequence for plasmid replication, ensuring multiple copies. Interplays with host replication machinery.	Ensures high copy number of plasmid, thus high gene dosage for GOI.	Plasmid cannot replicate; eventually lost from cells.	Impaired or no plasmid replication, leading to low copy number or loss of plasmid over generations.
- Antibiotic Resistance Marker (AmpR)	Gene for resistance (e.g., ampicillin), used for selection. Interplays with antibiotic to differentiate cells containing plasmid.	Allows selection of cells successfully transformed with the pET plasmid. Cells grow in antibiotic media.	Transformed cells cannot survive in antibiotic media; cannot be selected.	Non-functional resistance gene; cells cannot survive in selective media, failing to distinguish transformed cells.
- T7 Promoter	Strong and specific DNA sequence recognized exclusively by T7 RNA Polymerase. GOI is cloned downstream. Interplays with T7 RNA Polymerase for transcription initiation.	Ensures efficient recognition and transcription of GOI by T7 RNA Polymerase when active.	No T7 promoter: GOI cannot be transcribed by T7 RNA Polymerase.	If T7 promoter sequence mutated, T7 RNA Polymerase cannot bind effectively, leading to no/low transcription of GOI.
- lac Operator (lacO)	DNA sequence immediately downstream of T7 promoter, within the transcriptional start region. Binds lac Repressor to regulate transcription. Interplays with lac Repressor and IPTG.	Repressor binds lacO to block T7 promoter in absence of IPTG ('OFF' state).	No lacO: No repression by lac Repressor; constitutive expression (even without IPTG).	If lac operator sequence is mutated such that lac repressor cannot bind effectively, T7 promoter would be constitutively active, leading to uncontrolled, basal expression even without IPTG.
Host Cell Components	Provides the cellular machinery for transcription, translation, and regulation (e.g., BL21(DE3) strain).	Supports gene expression and protein synthesis. Optimized for recombinant protein production.	No host cell: no living system to express protein.	A non-compatible or non-functional host cell (e.g., lacking T7 RNA Pol) will yield no protein.
- T7 RNA Polymerase	A highly processive and specific enzyme (from T7 bacteriophage) that exclusively transcribes DNA downstream of the T7 promoter. Its gene is typically integrated into the host chromosome under a lacUV5 promoter. Interplays with T7 Promoter.	Upon induction (IPTG), efficiently transcribes GOI from T7 promoter, leading to massive mRNA and protein production.	No T7 RNA Polymerase to recognize the T7 promoter results in no transcription of GOI and no recombinant protein.	Non-functional T7 RNA Polymerase leads to no transcription of GOI, even with induction.
- lac Repressor (encoded by lacI gene)	Protein expressed from the host chromosome's lacI gene. Binds lac Operator to block T7 promoter. Interacts with lacO and IPTG.	Binds lacO, blocking T7 RNA Pol from T7 promoter in the absence of IPTG, maintaining the 'OFF' state and preventing toxic overexpression.	No functional lac repressor leads to constitutive activity of the T7 promoter and uncontrolled expression, potentially toxic.	Non-functional lac repressor protein cannot bind lacO effectively, leading to constitutive expression, likely affecting cell health.
Inducer
- IPTG	A synthetic, non-hydrolyzable analog of allolactose. Binds to lac Repressor, causing conformational change and its dissociation from lacO. Interplays with lac Repressor.	At proper concentration and timing, binds lac Repressor, leading to its dissociation from lacO, enabling T7 RNA Pol to bind T7 promoter and initiate transcription ('ON' state).	Without IPTG (or at too low conc.), lac Repressor remains bound to lacO, preventing efficient transcription and resulting in very low or no recombinant protein yield ('OFF' state).	A mutated IPTG that cannot bind lac repressor would fail to induce expression, keeping the system in the 'OFF' state.

Transcription in E. coli - Step-by-step breakdown

Initiation:
- The E. coli RNA polymerase, a multi-subunit enzyme, recognizes and binds to specific DNA sequences called promoters. Promoters typically consist of two conserved regions: the $-35$ region and the $-10$ region (Pribnow box).
- The binding of RNA polymerase causes the DNA double helix to locally unwind and open.
Elongation:
- RNA polymerase moves along the DNA template strand in the $3'$ to $5'$ direction.
- It synthesizes a complementary messenger RNA (mRNA) strand in the $5'$ to $3'$ direction, using ribonucleoside triphosphates (ATP, UTP, CTP, GTP) as building blocks.
Termination:
- Transcription stops when RNA polymerase encounters a terminator sequence on the DNA.
- These sequences often form a hairpin structure in the newly synthesized mRNA and/or involve a rho factor protein, which causes RNA polymerase to dissociate from the DNA and release the nascent mRNA.

Comparison of Prokaryotic vs. Eukaryotic Transcription

Feature	Prokaryotic Transcription (e.g., E. coli)	Eukaryotic Transcription
Definition	The process of synthesizing an mRNA molecule from a DNA template, occurring in the cytoplasm, often coupled with translation. Primarily performed by a single type of RNA polymerase.	The process of synthesizing an RNA molecule (pre-mRNA, rRNA, tRNA) from a DNA template. Occurs in the nucleus, separated from translation, and involves significant post-transcriptional processing. Carried out by three main types of RNA polymerases (Pol I, Pol II, Pol III) for different RNA types.
Pros	- Fast growth and easy to culture. - Low cost. - Simple genetics for easy manipulation. - Coupled transcription-translation for rapid protein production. - Efficient for high-yield recombinant protein expression with systems like pET.	- Allows for complex regulation and gene expression control. - Enables extensive post-transcriptional modifications (splicing, capping, polyadenylation) leading to protein diversity. - Offers compartmentalization for refined control over gene expression. - Suitable for expressing complex eukaryotic proteins with native-like PTMs in host cells.
Cons	- Lack of complex post-translational modifications (PTMs) (e.g., glycosylation, phosphorylation) found in eukaryotes. - Potential for protein misfolding and inclusion body formation. - Toxicity of some foreign proteins to the host cell. - Variance in codon usage can limit expression of eukaryotic genes.	- Slower and more energy-intensive process due to larger genomes and more complex regulatory elements. - Requires extensive post-transcriptional processing, adding complexity and time. - Multiple RNA polymerases and numerous transcription factors are needed, making the system more intricate. - Not directly coupled with translation, delaying protein synthesis compared to prokaryotes.
Functions	Produces mRNA, which is immediately available for translation, allowing for quick adaptation to environmental changes. Essential for all cellular processes.	Produces various RNA molecules (mRNA for protein synthesis, rRNA for ribosomes, tRNA for translation, snRNA for splicing, etc.) necessary for gene expression and cellular function. Enables fine-tuned regulation of gene expression and generation of protein isoform diversity through alternative splicing, critical for multicellular organisms and development.
Key Enzymes	Primarily one RNA polymerase (multi-subunit enzyme).	RNA Polymerase I (rRNA), RNA Polymerase II (mRNA, snRNA), RNA Polymerase III (tRNA, some rRNA). Each has specific promoters.
Location	Cytoplasm (no nucleus).	Nucleus.
mRNA processing	No significant post-transcriptional processing (no introns, no capping, no poly-A tail).	Extensive processing: - 5' capping: addition of a modified guanine nucleotide to the 5' end. - 3' polyadenylation: addition of a poly-A tail to the 3' end. - Splicing: removal of introns and ligation of exons.

Protein Expression: From Gene to Protein Review

I. Introduction & Goals

Overall Focus: Understanding how recombinant proteins are expressed in bacterial systems, purified, and analyzed for functional and structural properties.
Key Topics: Recombinant DNA techniques, protein expression, purification, characterization.
Emphasis this week: Lecture 8 outline (Why make/study proteins, Bacteria as factories, pET system).

II. Importance & Applications of Proteins

Role: Proteins are the "molecular machines" of the cell, driving virtually all cellular processes (e.g., actin/myosin for movement, hemoglobin for oxygen, Na+/K+ pump for electrical signals, opioid receptors for pain/mood).
Why produce in the lab? Required to understand:
- Structure: What it looks like.
- Function: What it does.
- Mechanism: How it carries out its function.
Applications: Used in research, industry, and therapeutics (preventing/treating diseases).

III. Native vs. Recombinant Protein Sources

Source	Definition	Pros	Cons	Preference
Native Protein	Isolated directly from natural biological sources (e.g., tissue, blood, cells).	Biologically active.	Low yield, Impure, Difficult to purify.
Recombinant Protein	Produced by introducing the gene of interest into a model organism (e.g., bacteria, yeast, mammalian cells).	Scalable, High yield, Consistent quality, Modifiable (e.g., via tags).	May require optimization for proper folding or activity.	Recombinant proteins are often preferred due to controlled, scalable production and ease of modification for research, industrial, or therapeutic applications.

IV. Expression Systems Overview

Choice depends on complexity, yield, and intended use.
Common systems: E. coli, Yeast, Insect Cells, Mammalian Cells (each with pros and cons).

V. Using E. coli as Protein Factories

Advantages:
- Fast growth and easy to culture.
- Low cost (media, equipment), high yield from small culture volumes.
- Simple genetics for easy DNA manipulation.

VI. Molecular Mechanisms in E. coli

A. Native Protein Expression in E. coli

Transcription:
- Carried out by RNA polymerase starting at the promoter and stopping at the terminator sequence.
Translation:
- Initiated when the ribosome binds to the ribosome-binding site (RBS) and recognizes start codon (AUG), terminating at the stop codon (e.g. UAA).

B. Recombinant Protein Expression in E. coli

Expression Plasmid: Contains the gene of interest, with key features:
1. Carries the gene of interest.
2. Inducible promoter controlling protein production.
3. Affinity tags that facilitate protein purification.

C. Limitations of Bacterial Expression Systems

Challenges:
- Lack of complex post-translational modifications (PTMs) (e.g., glycosylation, phosphorylation).
- Protein misfolding potential.
- Toxicity of some proteins to the host.
- Variance in codon usage between target gene and bacterial host.
Overcoming limitations: Possible to express eukaryotic proteins through proper vector design linking the eukaryotic gene to bacterial regulatory sequences.

VII. The pET Expression System

Name: Plasmid for Expression by T7 RNA polymerase.
Function: Facilitates high levels of recombinant protein production in E. coli.
Process: Gene of interest (GOI) is inserted into a pET vector, which is then introduced into E. coli for expression.

A. Key Components of the pET System

pET Vector: Contains instructions for both transcription and translation.
Host Cell: Provides the necessary protein machinery for transcription and translation processes.

B. The pET Vectors (Key Features)

Origin of replication (ORI): For plasmid replication.
Antibiotic resistance marker (AmpR): For selection of transformed cells.
T7 promoter: Linked to the gene of interest, controlling transcription initiation.
lac operator: For transcription regulation via the lac repressor, which blocks access to the promoter unless IPTG is present.

C. Mechanism of Action in the pET System

T7 Promoter & RNA Polymerase:
- T7 RNA Polymerase provides high transcription rates and specificity towards the T7 promoter.
- Distinct from E. coli RNA polymerase, T7 RNA polymerase recognizes a fixed 17 bp region rather than $-10/-35$ promoter regions.
Transcription Regulation:
- For transcription to occur, T7 RNA polymerase must bind to the T7 promoter, which is regulated by the binding state of the lac repressor and presence of the inducer IPTG.

D. Regulation of Transcription in the pET System

IPTG: A non-degradable analog of allolactose.
Mechanism: IPTG binds the lac repressor, causing it to dissociate from the lac operator and promote transcription of target genes by T7 RNA polymerase.

E. Inducible Protein Expression Process

Absence of IPTG (‘OFF’ State): Without IPTG, the lac repressor binds to lacO, blocking transcription.
Presence of IPTG (‘ON’ State): Presence of IPTG inactivates the lac repressor, allowing transcription to proceed.

F. Key Considerations for Protein Expression Timing

IPTG should be added when bacterial cultures reach mid-log phase growth (typically at OD600 = $0.6-0.8$ in LB media). This timing ensures sufficient biomass for high protein production.

G. Advantages of the pET System

High yield of proteins due to efficient T7 RNA polymerase.
High specificity of T7 promoters guarantees focused transcription.
Controlled expression through IPTG, along with enhanced purification ease via incorporation of tags like His-tag for affinity chromatography.

VIII. E. coli Transcription Step-by-step breakdown

Initiation:
- The E. coli RNA polymerase, a multi-subunit enzyme, recognizes and binds to specific DNA sequences called promoters. Promoters typically consist of two conserved regions: the $-35$ region and the $-10$ region (Pribnow box).
- The binding of RNA polymerase causes the DNA double helix to locally unwind and open.
Elongation:
- RNA polymerase moves along the DNA template strand in the $3'$ to $5'$ direction.
- It synthesizes a complementary messenger RNA (mRNA) strand in the $5'$ to $3'$ direction, using ribonucleoside triphosphates (ATP, UTP, CTP, GTP) as building blocks.
Termination:
- Transcription stops when RNA polymerase encounters a terminator sequence on the DNA.
- These sequences often form a hairpin structure in the newly synthesized mRNA and/or involve a rho factor protein, which causes RNA polymerase to dissociate from the DNA and release the nascent mRNA.

IX. Comparison of Prokaryotic vs. Eukaryotic Transcription

Feature	Prokaryotic Transcription (e.g., E. coli)	Eukaryotic Transcription
Definition	The process of synthesizing an mRNA molecule from a DNA template, occurring in the cytoplasm, often coupled with translation. Primarily performed by a single type of RNA polymerase.	The process of synthesizing an RNA molecule (pre-mRNA, rRNA, tRNA) from a DNA template. Occurs in the nucleus, separated from translation, and involves significant post-transcriptional processing. Carried out by three main types of RNA polymerases (Pol I, Pol II, Pol III) for different RNA types.
Pros	- Fast growth and easy to culture. - Low cost. - Simple genetics for easy manipulation. - Coupled transcription-translation for rapid protein production. - Efficient for high-yield recombinant protein expression with systems like pET.	- Allows for complex regulation and gene expression control. - Enables extensive post-transcriptional modifications (splicing, capping, polyadenylation) leading to protein diversity. - Offers compartmentalization for refined control over gene expression. - Suitable for expressing complex eukaryotic proteins with native-like PTMs in host cells.
Cons	- Lack of complex post-translational modifications (PTMs) (e.g., glycosylation, phosphorylation) found in eukaryotes. - Potential for protein misfolding and inclusion body formation. - Toxicity of some foreign proteins to the host cell. - Variance in codon usage can limit expression of eukaryotic genes.	- Slower and more energy-intensive process due to larger genomes and more complex regulatory elements. - Requires extensive post-transcriptional processing, adding complexity and time. - Multiple RNA polymerases and numerous transcription factors are needed, making the system more intricate. - Not directly coupled with translation, delaying protein synthesis compared to prokaryotes.
Functions	Produces mRNA, which is immediately available for translation, allowing for quick adaptation to environmental changes. Essential for all cellular processes.	Produces various RNA molecules (mRNA for protein synthesis, rRNA for ribosomes, tRNA for translation, snRNA for splicing, etc.) necessary for gene expression and cellular function. Enables fine-tuned regulation of gene expression and generation of protein isoform diversity through alternative splicing, critical for multicellular organisms and development.
Key Enzymes	Primarily one RNA polymerase (multi-subunit enzyme).	RNA Polymerase I (rRNA), RNA Polymerase II (mRNA, snRNA), RNA Polymerase III (tRNA, some rRNA). Each has specific promoters.
Location	Cytoplasm (no nucleus).	Nucleus.
mRNA processing	No significant post-transcriptional processing (no introns, no capping, no poly-A tail).	Extensive processing: - 5' capping: addition of a modified guanine nucleotide to the 5' end. - 3' polyadenylation: addition of a poly-A tail to the 3' end. - Splicing: removal of introns and ligation of exons.

X. Important Reminders and Upcoming Assessments

Quiz #4 this Friday. Review LockDown browser on D2L. One-page cheat sheet and calculator allowed. Phones away.