Cell-Free Protein Expression Using a Bacterial Crude Cell Extract

A cell-free protein expression system uses a bacterial crude cell lysate, which contains the molecular machinery essential for in vitro transcription and translation.

This lysate includes transcription factors, ribosomes, and transfer RNAs (tRNAs).

Add a mix containing a DNA template, the RNA polymerase, nucleotides, and amino acids to the lysate.

Transfer it to a PCR plate and centrifuge to collect the liquid sticking to the walls. Seal the plate to prevent evaporation.

Incubate in a thermal cycler at an optimal temperature to support cell-free protein expression.

The transcription factor associates with the RNA polymerase to form a complex that binds to the template DNA.

This complex catalyzes RNA synthesis, incorporating nucleotides to generate a messenger RNA (mRNA).

The mRNA recruits ribosomes, which initiate translation, during which tRNAs deliver the corresponding amino acids to form a polypeptide chain.

The polypeptide folds into a functional protein, ready for downstream applications.

To perform cell-free protein expression using DNA template thaw 10X energy solution master mix, 4X amino acid master mix aliquots, and DNA template at room temperature. Then remove the T7 RNA polymerase and RNAse inhibitor stocks from the minus 20 degrees Celsius freezer and place on ice. Once the DNA template, 10X energy solution master mix, and 4X amino acid master mix are thawed, place on ice.

Prepare a cell-free reaction master mix by adding each component in the accurate order to a two-milliliter tube on ice, and gently flick the tube after each addition to the master mix. Remove Vibrio natriegens crude cell lysate from the minus 80 degrees Celsius freezer and place on ice for 10 to 20 minutes until thawed. Add the appropriate volume of thawed crude cell extract to the cell-free reaction master mix and gently mix using a pipette.

To perform endpoint cell-free protein expression using thermocycler, first add 10 microliters of the cell-free reaction master mix per well of the bottom of a 96-well PCR plate making sure to mix the master mix by flicking the tube gently in between each transfer to the PCR plate. Centrifuge the plate at 1, 000 times g for 10 seconds to pool any master mix that may have been stuck to the sides of the wells. And then seal the wells with a plate adhesive to prevent evaporation.

Place the plate into a thermocycler set at 26 degrees Celsius with a heated lid set at 105 degrees Celsius. After incubating the reactions in the PCR plate for a minimum of three hours, expressed proteins can be purified, quantified and used for downstream processes.