Recombinant Proteins

Expression of Recombinant Proteins

• Modern Methods in Protein Research

Utility of Recombinant Proteins

• Applications:

  • Analysis of activity

  • Study of structure-function relationship

  • Development of antibodies

  • Production of enzymes for biotechnology

  • Synthesis of proteins for medical applications

  • Creation of vaccines

Selection of Expression Systems

• Considerations for choosing an expression system include:

  • System Size

  • Proteolytic Cleavage: Applicable only in mammalian and insect systems

  • Purity/Yield:

  • E. coli: >50%

  • Mammalian: <1%

  • Insect: >30%

  • Yeast: ~1%

  • Amounts Needed:

  • Functional studies: micrograms (μg)

  • Antibody production: milligrams (mg)

  • Biotechnology applications: grams-kilograms (g-kg)

  • Protein State:

  • Native (active) vs. denatured (epitopes exposed)

  • Post-translational Modifications: Required or not

  • Codon Preference: Sequence optimization for expression

  • Cost and Speed: Economic considerations

  • System Availability: Accessibility and investment needed for testing

Steps in Making Recombinant Proteins

1. Clone or synthesize the Open Reading Frame (ORF) of the desired protein

2. Clone the ORF into an expression vector

3. Transform (transfect) target cells with the vector

4. Grow the cells and induce protein expression

5. Purify the expressed protein

• Note: The expression and purification process can be complex/challenging

Protein Expression Requirements

• Key Requirements:

  • Presence of a strong promoter for effective initiation of transcription

  • Efficiency of translation mechanisms

  • Need for post-transcriptional modifications (if required)

  • Stability of the expressed protein for activity

Promoters in E. coli

• Definition: Sequences that RNA polymerase binds to initiate transcription.

• Types Used:

  • E. coli RNA polymerase or T7 phage RNA polymerase

E. Coli RNA Polymerase Promoters

• Key Elements:

  • Two essential regions in E. coli genes:

  • -35 bp region (Consensus: TTGACA)

  • -10 region (Pribnow box, Consensus: TATAAT)

  • Transcription Start Site: +1

Specific Promoters Identified:

• lac: GGC TTTACA (18 nt)

• trp: CTG TTGACA (17 nt)

• tac: CTG TTGACA (17 nt) - 11X stronger than lac

• Consensus: TTGACA (17 nt) TATAAT

Translation in E. coli

• Shine-Dalgarno Sequence: GGAGG

  • Critical for ribosome binding and initiation of translation

• Effects on Translation Efficiency:

  • Degree of complementarity to 16S rRNA

  • Distance from the promoter to the Shine-Dalgarno sequence (ideally 50 bp from AUG)

  • Ensuring accessibility of the sequence for ribosomal binding (not folded in secondary structure)

Codon Choice in Protein Expression

• Synonymous Codon Usage: Not all synonymous codons are used equally, depending on tRNA efficiency and availability.

• Impact on Expression:

  • Presence of rare codons (e.g., AGA, AGG) may reduce expression levels

  • High GC content can also decrease protein expression

Benefits of Making Fusion Proteins

• Advantages:

  • Convenient for isolation and purification of proteins

  • Enhanced detection capabilities

  • Targeting to specific cellular compartments

  • Protection against proteolysis

  • Improvement in solubility of proteins

Advantages and Disadvantages of Fusion Proteins

Advantages: Tagged Proteins

• Simplified purification via generic protocols

• Enhanced detection of target proteins

• Improved solubility and stability

• Ability to incorporate targeting information into fusion tags

• Marker for protein expression

• Some tags: strong binding to chromatography media in presence of denaturants, on-column refolding is possible.

Disadvantages:

• Potential interference with protein structure from tags —> affect folding and biological activity

• Difficulty in complete tag removal, which may leave residual amino acids if cleavage not 100%

• Less straightforward purification and detection processes

Fusion Protein Tags

• Common Tags:

  • Poly-His: 6 residues, sequence: HHHHHH, used for affinity purification

  • Glutathione S-Transferase (GST): used for purification via cross-linked amylose

  • Streptavidin-binding peptide: binds strongly to streptavidin for purification

  • Various tags like c-myc, HA, T7 for detection using antibodies

Disulfide Bond Formation in Fusion Proteins

• For some proteins: formation of correct disulfide bonds is required for folding in native conformation and enhanced solubility

• Challenge: Cytosol of E. coli has reducing properties —> disulfide bonds not formed —> formation of inclusion bodies

• Solution: Transporting proteins (translocation) to periplasmic space enables oxidative conditions for disulfide bond formation and improved yield.

• Also protects from proteolysis and helps purification

• Recombinant protein translocation achieved by fusing them to periplasmic proteins.

Recombinant Protein Purification Techniques

• Extraction Processes:

  • Gentle Methods: E.g., osmotic shock, enzymatic digestion for cell lysis

    • Low product yield but reduced protease release. Lab scale only, mechanical disruption.

  • Typical Conditions: Glass beads, freeze/thaw cycles

    • Physical method. Several cycles.

  • Vigorous Techniques: Ultrasonication, bead milling, French press homogenization for larger scale

    • Ultrasonication/bead milling: small scale, release of NA may cause viscosity problems, inclusion bodies must be resolubilized.

  • Commentary: Balance yield vs. purity in varying conditions

Factors Affecting Protein Solubility in Fusion Proteins

• Influencing Factors:

  • Size of the expressed protein

  • Nature of the expressed protein (e.g., transcription factor, enzyme)

  • Level of expression and choice of expression system

  • Nature of the fusion tag used

Effects of Tag Size on Solubility

• Research Findings:

  • Studies show varying effects of different fusion tags on solubility and purification yield of test proteins

  • Important to select optimal tags based on specific protein requirements for successful expression and purification.