Recombinant Proteins

Native vs. Recombinant Proteins

• Abundant Protein Sources: To study or utilize a protein, a relatively abundant source is needed.

  • Types of Sources:

    • Native Proteins:

    • Isolated from natural sources, such as plant or animal tissues.

    • Recombinant Proteins:

    • Produced via recombinant DNA technology.

• Methods for Producing Recombinant Proteins:

  • Homologous Expression:

    • Production of a protein in the same cell type from which it originates.

    • Example: Overexpression of an E. coli protein in an E. coli cell.

  • Heterologous Expression:

    • Production of a protein in a cell type that does not naturally produce that protein.

    • Example: Overexpression of a jellyfish protein in an E. coli cell.

Advantages of Recombinant Proteins

• Creation of an abundant source of the protein of interest.

  • Many native proteins produced at very low concentrations.

  • Recombinant proteins can be over-expressed, i.e., produced at high concentrations.

• Safety:

  • Many useful native proteins may come from dangerous or pathogenic sources (e.g., blood, snakes).

  • Recombinant proteins can be produced in non-pathogenic, non-toxic hosts, mitigating safety risks.

• Alteration of Amino Acid Sequence:

  • Molecular biology techniques allow alteration of source DNA coding for the protein.

  • Enables amino acid omission/substitution, useful for creating more stable or effective proteins, or for fusion proteins (multiple proteins joined together).

Examples of Recombinant Proteins on the Market

• Adapted from: Protein Biochemistry and Biotechnology, Gary Walsh.

Importance of Native Protein Production

• Natural Overexpression:

  • Some proteins are naturally overexpressed and thus easier and cheaper to purify.

  • Example: Human serum albumin naturally present at 42 g/L, which current recombinant systems cannot match.

• Functionality Issues:

  • Some proteins are difficult to produce in recombinant form and require the native source for functional integrity, due to:

  • Poor folding in hosts or toxicity.

  • Specific and important post-translational modifications (PTMs) may not be reproducible in host cells.

• Patents and Public Perception:

  • Some recombinant proteins may be patented.

  • Public suspicion regarding recombinant products as “genetically modified,” leading companies to favor native proteins.

Examples of Native Commercial Proteins

Creation of a Recombinant Protein

• General Steps: Taken from Protein Biochemistry and Biotechnology, Gary Walsh.

• Detailed Look at the Process: Refer to actual detailed documentation for specific steps mentioned in class.

The Central Dogma of Molecular Biology

• Components:

  • DNA → RNA → Proteins

  • Process Steps:

  • Transcription

  • Translation

  • Replication

Expression Hosts for Protein Production

• Various systems exist for recombinant protein production, ranging from prokaryotic to eukaryotic sources.

Prokaryotic and Simple Eukaryotic Systems

• Common Systems:

  • E. coli

  • Yeast: Such as Saccharomyces cerevisiae and Pichia pastoris

  • Fungi: Particularly from Aspergillus species.

Eukaryotic Expression Systems

• Animal Cells:

  • Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cells.

• Transgenic Animals:

  • Sheep and goats producing human proteins in milk (Example: Tracy, the transgenic sheep used to produce Alpha1 antitrypsin).

• Plant-Based Systems:

  • E.g., tobacco plant (Nicotiana tabacum).

• Insect Cell Culture:

  • Using viral transfection with baculovirus (e.g., Sf9 and Sf21 cells from Spodoptera frugiperda).

Sources of Biopharmaceuticals

• Majority of biopharmaceuticals on the market are recombinant proteins.

• Therapeutic recombinant proteins:

  • Approved to date predominantly produced in:

  • E. coli, S. cerevisiae (yeast), or animal cell lines (mainly CHO or BHK cells).

  • In research purposes: other fungi, insect cells, transgenic plants, and animals.

Protein Expression in Prokaryotes

• Definition: Process of protein production by host cells which provides the machinery for protein synthesis.

  • Genetic Elements Needed:

  • Promoter

  • Control sequences

  • Transcriptional start and stop sequences

  • Ribosome Binding Sites (RBS)

  • Translational start and stop codons.

  • Cloning vectors lack transcriptional and translational elements, necessitating use of expression vectors.

  • Various E. coli expression vectors discussed.

Expression Vectors: Basic Constructs

• Components Include:

  • Origin of Replication (ori): Facilitates cloning and amplification of plasmids.

  • Selection Marker: Indicates successful uptake of vector (e.g., antibiotic resistance).

  • Expression Cassette: Contains:

  • Promoter

  • Ribosome Binding Site

  • Fusion Tag

  • Cleavage Site

  • Multiple Cloning Site (MCS)

  • Terminator Region.

Origin of Replication (Ori)

• Definition: DNA sequence that initiates replication within a plasmid.

• Example Plasmids and Their Copy Numbers:

  • pMB1: 15-60 copies/cell

  • pUC: 500-700 copies/cell

  • ColE1: 15-20 copies/cell.

• High copy number not always beneficial; excessive plasmids may incur metabolic burden, reducing protein yield.

Synthetic Operons and Promoters

• Synthetic Operon Requirements:

  • Repressor gene sequence

  • Promoter region

  • Operator region

  • Transcriptional start site

  • Ribosome Binding Site

  • Gene of interest with stop/start codons

  • Transcriptional termination sequence.

Promoters and Their Functionality

• Promoter Types:

  • Genes can be expressed constitutively or be switched on/off.

  • Plasmid promoters orchestrate expression and transcription of the gene of interest.

  • Promoters hold RNA polymerase binding sites and binding sites for transcription factors ( enhance/inhibit transcription).

• Various types available depending on host (bacterial, yeast, mammalian) and genetic constructs.

Promoter Strength and Inducers

• Inducible Expression: Promoters can be enhanced or suppressed by external inducers.

• Strong promoters typically lead to more substantial mRNA and protein expression.

• Inducers can be added/removed to control protein expression levels.

Classical Bacterial Promoters

• Lac Operon-Based Systems: Prominent source of bacterial promoters used in recombinant systems.

The Lac Promoter

• The Lac Promoter is vital and commonly used in recombinant DNA technology.

• Induction occurs through the presence of non-hydrolysable lactose (IPTG) that can bind to the Lac repressor protein.

LacUV5 Promoter

• This promoter variant is not sensitive to catabolite repression, ensuring effective transcription even under high glucose conditions.

Problems with Lac/LacUV5 Promoters

• Leaky Expression: Both promoters experience low-level expression even without inducer.

• A mutated lacI gene called LacIQ results in a tenfold increase in expression, offsetting leaky expression.

Tac Promoter

• A hybrid promoter combining sequences from both the trp and lac promoters, balancing strengths for optimal protein overexpression.

• Commonly used in commercial vectors such as pMAL.

T7/Lac UV5 System

• Renowned system found in pET vectors for high-level expression of target proteins.

• Can produce protein levels up to 50% of total E. coli cell content.

T7-Lac Promoter System Interaction

• Requires introducing the T7 polymerase gene, potentially via a plasmid or chromosomal integration, to facilitate high-level expression.

AraC-pBAD System

• The Arabinose Operon (araBAD) manages transcription through an activator protein that binds to arabinose, influencing gene expression depending on arabinose availability.

pBAD Vectors

• These vectors integrate necessary operon components and work with E. coli strains that can transport arabinose but not metabolize it.

Summary of Promotor Types

• Key Points:

  • Lac promoter: IPTG-inducible but weak.

  • Lac UV5: Improved IPTG-induced performance with better repressor production.

  • Trp promoter: Stronger than Lac promoter.

  • T7/Lac: Uses T7 polymerase and promotes significant overexpression.

  • AraBAD: Arabinose-inducible, minimizing leaky expression.