Month: July 2020

A common problem when expressing proteins recombinantly is that eukaryotic proteins are often insoluble when expressed in bacterial systems.

In this post we bring you some tips to establish the conditions to optimize the solubility of the proteins that are recombinantly expressed.

1.- SELECT THE APPROPRIATE TAG

Some tags, in addition to simplifying the protein purification process, can help improve its solubility.

Among the tags that contribute to increasing the solubility of proteins , we can highlight the tail of GST and MBP, which on the other hand can be a drawback due to their large size.

2.- IPTG CONCENTRATION

IPTG (isopropyl β-D-1-thiogalactopyranoside) is added during the production process of recombinant proteins to induce their expression .

When seeking to increase the solubility of proteins, it is not convenient to saturate cellular folding mechanisms, so reducing the concentration of IPTG can be beneficial in certain cases.

3.- INDUCTION TEMPERATURE

Solubility generally improves at temperatures below 37ºC . By reducing the temperature, the speed of protein production is reduced, which can contribute to increasing its solubility.

4.- INDUCTION TIME

Too long induction times can impair the solubility of proteins. It is advisable to take samples every few hours to test the expression, bearing in mind that the induction times must be greater the lower the temperature.

5.- CULTURE MEDIUM

Certain factors that improve solubility can be added to the culture medium:

• Prosthetic groups or cofactors necessary for the correct folding and stability of the protein.
• Buffers to avoid fluctuations in pH levels.
• Polyalcohols or sucrose that, by increasing the osmotic pressure, can help stabilize the protein’s structure.

6.- CO-EXPRESSION

Another option is to express the protein of interest together with proteins that can increase soluble protein levels, such as chaperones .

7.- SEARCH FOR ALTERNATIVES

• Small domains are generally more soluble than complete proteins, so one of the alternatives is to try to express only part of the protein of interest.
• Use alternative expression systems such as baculovirus or mammalian cells.
• Purify the protein under denaturing conditions and then proceed to its refolding.

When storing biological samples that we will later use in our research project, the selection of the most appropriate temperature to do so depends on factors such as the type of biological material, the solution in which it is suspended, the application of the sample or the expected storage time, among others.

In this post we bring you a summary of the most common storage temperatures for storing biological samples .

1.- STORAGE AT ROOM TEMPERATURE (15ºC / 27ºC)

Storage at room temperature has the advantage that the sample will be available for immediate use, and the costs and resources associated with the storage infrastructure are less than in the case of cold systems.

However, only some samples are suitable to be stored under these conditions. The biological samples that can be stored at room temperature include:

• Lyophilized samples.
• Paraffin-embedded fabrics.
• Saliva samples for genomic DNA studies with the appropriate preservative .
• Samples with preservatives that allow their stability at room temperature.
• Biological fluids for short periods of time from their obtaining (2-4h).

2.- REFRIGERATION (4ºC / 8ºC)

Refrigeration of biological samples is a good solution for short-term storage of samples that are stable at temperatures between 4 and 8ºC.

They can be stored refrigerated:

• Biological reagents such as enzymes or antibodies that are used frequently.
• Biological liquids previously centrifuged, up to a maximum of 24h.

3.- FREEZING (-20ºC)

Freezing is the technique of choice for storing biological samples for short periods of time, in those cases where they are not stable at higher temperatures.

They are usually stored at -20ºC:

• Nucleic acids (DNA / RNA).
• Plasma samples, up to a maximum of 4 weeks.
• Aliquoted biological reagents that are expected to be used in the short term.

4.- DEEP FREEZING (-80ºC)

Deep freezing at -80ºC is the method of choice for storing long-term biological samples, since it prevents the degradation of nucleic acids, proteins and other biomolecules.

Among the samples that are stored deep-frozen are:

• Tissues and organs.
• Nucleic acids (DNA / RNA) that are not expected to be used in the short term.
• Cells.
• Plasma samples.

5.- CRYOGENIZATION (-150ºC / -190ºC)

Cryogenization is the gold standard for storing biological samples for long periods of time. When stored at such low temperatures (<-135ºC), the biological activity in the sample is completely suspended, avoiding degradation processes.

Cryogenization is usually the method of choice for long-term preservation of:

• Tissues and organs.
• Cells.