Untitled Quiz

Study Notes

Lab 3: Recombinant Protein Expression

Host organism: Escherichia coli is the preferred host for initial recombinant protein production, regardless of the protein's origin. Specific strains like BL21 (DE3) pLysS + RIL are used.

IPTG Induction Protocol

T7 RNA polymerase system: This system is highly effective for producing recombinant proteins. The T7 RNA polymerase gene is inserted into the bacterial chromosome and is controlled by the inducible lac UV5 operon. It's transcribed by the host's cellular polymerase.
Lac repressor protein (LacI): Controls access to the T7 RNA polymerase gene by binding to the lac UV5 operon.
IPTG (isopropyl-β-D-1-thiogalactopyranoside): An inducer that triggers protein expression by acting as a structural analogue to allolactose. IPTG is the currently most efficient method. It causes the T7 RNA polymerase to transcribe the desired protein's coding sequence.
T7 promoter: The plasmidic T7 promoter controls the transcription of the protein of interest, allowing regulation through the lac operator (T7 lac promoter).

Protocol

Overnight culture: Start with a fresh bacterial culture (less than 4 weeks old). Grow the colony overnight at 37°C in 5ml LB broth with ampicillin (AMP) in a 15ml falcon tube on a shaker incubator (150-200 RPM).
Optical Density (OD): Monitor bacterial growth by measuring OD600, to ensure the culture is in the exponential (log) phase (OD600: 0.6-0.8) for optimal induction.
Induction: Remove a 1ml portion of the bacteria and add a 100 µl 100mM IPTG solution to 9 ml of the existing bacterial culture (This adjusts the solution to a final volume of 10ml and a concentration of 1mM IPTG). Maintain at 37°C with shaking (150-200 RPM) for 30 minutes and various intervals (1, 2, 3, and 24 hours) to observe the effect of the induction time. Take samples from the induced culture and those that were not for control at these various points for SDS-PAGE analysis. Freeze these at -20°C until needed.
Total Cell Protein Preparation for SDS-PAGE: Add 100 µl sample buffer (SDS loading buffer) to uninduced or IPTG-induced samples. Heat samples at 95°C for 10 minutes and centrifuge at 14,000 RPM for 5 minutes.
SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis): A technique that separates proteins based on their molecular weight. The gel has stacking (4%) and separating (10% or 12%) gels. Use a pre-stained protein marker in one lane to determine molecular weights.
Coomassie Blue Staining: The separated proteins are visualized using Coomassie blue staining. After electrophoreses and destaining, photograph the gel.

SDS-PAGE Gel Preparation

Gel components: Acrylamide, bis-acrylamide, ammonium persulphate, TEMED, and appropriate buffer solutions for differing gel concentrations (various ratios of acrylamide to provide different molecular weight ranges) are combined for the separating and stacking gels.
Polymerization: Allow the gel to solidify and form wells. The gel should be clearly formed.
Protein Loading: Load protein samples into the gel wells. The volume loaded depends on factors such as sample size and the desired staining intensity.
Electrophoresis: Apply an electrical current to separate proteins.
Staining and Visualization: Stain the proteins and visualize them.
Gel Documentation: Document and analyze the gel.