mmg 2040 lecture 27 - recombinant protein expression

Define: transcriptome, proteome, translatome, isoelectric focusing

transcriptome: Set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA, produced in one or a population of cells

proteome: Total set of proteins encoded by a genome or the total protein complement of an organism

translatome: total set of proteins that have actually been translated and are present in a cell under any particular set of conditions

isoelectric focusing: Technique for separating proteins according to their charge by means of electrophoresis through a pH gradient

• high-resolution electrophoretic technique that separates proteins or amphoteric molecules based on their unique isoelectric point —> pH where they carry no net charge

• Molecules migrate through a stable pH gradient under an electric field until they reach a zone where the pH equals their pi, stopping because they become neutral

• Principle of Isoelectric Focusing 

  • 𝒑𝑰 (Isoelectric Point): The specific pH value where a molecule's net charge is zero.

  • pH Gradient: IEF utilizes a gel or medium with an immobilized, established pH gradient (acidic to basic).

  • Migration: When an electric field is applied, molecules with a positive net charge move toward the cathode, and negatively charged molecules move toward the anode.

  • Focusing: As molecules migrate, they reach the pH corresponding to their 𝑝𝐼, lose their net charge, and stop moving. This "focuses" them into sharp bands

Provide reasons for the discrepancy between: proteome and translatome, proteome and transcriptome

Some RNA molecules are non-coding

Alternative splicing →multiple protein products

Levels of mRNA may not correlate with protein levels due to differential rates of mRNA translation or degradation

Regulation of proteins by addition or removal of acetyl, phosphate, AMP, ADP-ribose, or other groups

Regulation of proteins by chemical modification of amino acid residues

Modification by proteolytic cleavage or addition of sugar or lipid residues to give glycoproteins or lipoproteins

Proteins themselves may be degraded and vary greatly in stability

Identify the essential components of an expression vector used for eukaryotic expression systems

• Strong promoter (e.g., T7)

• Ribosome binding site

• Selectable marker

• Tag sequence (His-tag, GFP)

• PolyA site

Describe the basics steps in recombinant protein expression

1. Clone gene into expression vector

• Expression Vectors

• Strong promoter (e.g., T7)

• Ribosome binding site

• Selectable marker

• Tag sequence (His-tag, GFP)

• PolyA site

2. Introduce into host cell

• Bacterial systems - Heat-shock transformation + Electroporation

• Yeast systems - Heat-shock transformation + Electroporation

• Mammalian systems - Calcium phosphate transfection + Lipid-mediated transfection + Viral Transduction

3. Induce expression (using lacuv, t7, tet-on/off)

• Host produces protein

• Control timing of expression

• Prevents toxicity

5. Harvest cells - General Purification Strategy

• Lyse cells

• Separate proteins

• Isolate target protein

• Validate purity

Compare pros/cons of bacterial expression systems

bacterial: simple production, low cost, efficient production

• lacks post-translational machinery

• protein expressed in inclusion bodies

• presence of endotoxins

Compare pros/cons of insect expression systems

insect: Efficient, native post-translational modification of complex proteins, baculovirus is not harmful to humans, livestock, or poultry

• cells eventually die —> non-continuous expression

• new infection for each new round of production

• toxicity of transfection agents

• complex procedures

Compare pros/cons of mammalian expression systems

mammalian: best post-translational modifications to native protein, regulation + inducible expression

• complex procedures, high cost, viral vectors can infect several hosts

Compare/contrast transient and stable transfections

transient: not integrated in genome but remains in nucleus, not passed onto progeny/genetic alteration is not permanent, does not require selection, high copy number of material results in high level of protein expression, generally not suitable for studies using inducible vectors

• both DNA/RNA vectors can be used

• harvested 24-96 hrs after transfection

stable: integrated into genome carried stably from gen. to gen./genetic alteration is permanent, requires selective screening for stable transfectants, single/low copy number of integrated

stable vs. transient transfection when entering cells

Define transfection and transduction relative to mammalian expression vectors

transfection: Introduction of vectors, without using viruses, into mammalian cells

Transduction: Introduction of vectors into mammalian cells using viral-mediated delivery

Design a basic mammalian expression strategy

Bacterial or mammalian system?
Transient or stable?
Inducible or constitutive?
Viral or non-viral delivery?

a cloning experiment - it is important it is IN FRAME

Describe the following inducible promoter systems: lacUV

The lacUV5 promoter is a mutated promoter from the Escherichia coli lac operon very similar to the classical lac promoter,

• it requires no additional activators and it drives high levels of gene expression

The lacUV5 promoter sequence conforms more closely to the consensus sequence recognized by bacterial sigma factors than the traditional lac promoter does

• Due to this, lacUV5 recruits RNA Polymerase more effectively

  • leads to higher transcription of target genes

• lacUV5 works independently of activator proteins or other cis-regulatory elements

• While no activators are required, lacUV5 promoter expression can be regulated by the LacI repressor and can be induced with IPTG, which is an effective inducer of protein expression

  • Due to this control, the lacUV5 promoter is commonly found on expression plasmids

  • is used when controllable but high levels of a product are desired

Describe the following inducible promoter systems: T7

T7 expression vectors (commonly pET vectors) enable high-level protein production in E. coli by using T7 RNA polymerase to specifically transcribe a target gene

• system is induced by IPTG, which triggers the expression of T7 RNA polymerase from the host genome

• recognizes a T7 promoter on the plasmid to drive massive target gene expression

• Vector Construction: The target gene is inserted into a plasmid (e.g., pET) downstream of a strong T7 promoter.

• Host Cell: The vector is introduced into special E. coli strains that have the gene for T7 RNA polymerase integrated into their chromosome.

• Induction: T7 RNA polymerase expression is controlled by an inducible promoter (typically lacUV5). The addition of IPTG relieves repression, producing T7 RNA polymerase.

• Transcription: The produced T7 RNA polymerase specifically transcribes the target gene, leading to high-level protein overexpression.

• Tight Regulation: To prevent basal expression (leakiness) of potentially toxic proteins, T7 lysozyme (a natural inhibitor) can be expressed, or the system can use Lac repressor binding

Describe the following inducible promoter systems: Tet-on/Tet-off

Tet-Off and Tet-On systems are inducible gene expression tools that use tetracycline or its derivative doxycycline (Dox) to control gene activity.

• both relying on a transactivator protein binding to a Tetracycline Response Element (TRE) promoter

Tet-Off turns expression off when Dox is added

• requires tTA (Tet-Off): Tetracycline-controlled Transactivator

• The tTA protein binds to the TRE promoter, activating transcription of the target gene

• Dox binds to the tTA protein, preventing it from binding to the TRE. Expression turns OFF

• Useful for long-term studies where expression is usually needed, then turned off

Tet-On turns expression on when Dox is added

• requires rtTA (Tet-On): Reverse Tetracycline-controlled Transactivator

• The rtTA protein cannot bind the TRE. Expression is OFF

• Dox binds to the rtTA protein, inducing a conformational change that allows it to bind the TRE. Expression turns ON

• Often preferred for rapid response to Dox induction