Soluble Protein Test Introduction and Method

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins.
Soluble protein definition from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC544838/
The soluble protein test procedure is based on attempting to dissolve chemicals in various solvents with a increasingly rigorous mechanical techniques. The solvents to be used, in the order of preference, are cell culture media, DMSO, and ethanol. Solubility shall be determined in a step-wise procedure that involves attempting to dissolve a test chemical in the solvents (in the order of preference) at relatively high concentrations using the sequence of mechanical procedures. If the chemical does not dissolve, the volume of solvent is increased so as to decrease the concentration by a factor of 10, and then the sequence of mechanical procedures are repeated in an attempt to solubilize the chemical at the lower concentrations.

Soluble protein test introduction from: https://ntp.niehs.nih.gov/

Test Method For Solubility Determination:

1. Take the remaining tube of induced cells and resuspend in 50 uL of B-PER containing protease inhibitors (PMSF or Complete).
2. Incubate at room temperature for 10 mins.
3. Spin down in a microcentrifuge at maximum speed for 10 min at 4 ºC.
4. Carefully transfer all of the supernatant into a new microfuge tube. Add 50 uL of 2x SDS-PAGE buffer. This is the soluble fraction.
5. Resuspend the pellet in 100 uL of 1x SDS-PAGE buffer. This is the insoluble fraction.
6. Boil the samples for 10 min, then cool down to room temperature.
7. Centrifuge for 5 mins at maximum speed at room temperature.
8. Analyze 15 uL of each sample using SDS-PAGE, with western blotting if necessary.

This soluble protein test protocol is for proteins expressed under the control of the lac, tac, or T7 promoters.

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