Determining the Optimal Temperature of Protein Expression
Without any doubt, one of the greatest advances in molecular biology is the development of simple and easy to carry out protocols for the expression of proteins from Escherichia coli cells transformed by a plasmid. These plasmids are synthesized in the lab and typically contain three major regions of interest: an antibiotic resistance marker (for selection of successfully transformed cells), a lacZ promoter (for activation of a gene of interest upon addition of lactose), and a gene corresponding to the protein of interest. While other cell lines (and even cell-free systems) are capable of taking up plasmids to express a protein of interest, E. coli is most widely used because of its fast growth and high transformation efficiency. Even though recombinant protein expression is a relatively recent development, its rapid expansion into all facets of molecular biology and biochemistry has led to a remarkable standardization into the optimal expression conditions for E. coli. However, one should be aware of the purposes of these conditions; and the potential benefits of deviating from them.
One such condition that researchers seldom give any thought to is the temperature at which to express E. coli after induction. The vast majority of protocols call for expression to be carried out at 37℃ for anywhere from 2-4 hours. Since the optimal temperature for growing E. coli is 37℃, it makes sense that this is considered the default. While expressing at 37℃ is ideal for many proteins, it can introduce issues for others. For this reason, there are a myriad of benefits to expressing at lower temperatures between 10℃ – 15℃. The most significant benefit to a low-temperature expression is an increase in the solubility of the expressed protein. This can be due to many factors. For example, lower temperatures greatly increase the time proteins have to fold, which reduces overall protein aggregation. This factor in particular aids the expression of larger proteins, or those with highly complex folding processes. Another reason to express at lower temperatures is the increase of properly folded proteins. One major drawback to E. coli is that the rates of transcription and translation are not optimized for all proteins, which reduces the overall time that newly expressed proteins spend in bacterial chaperones, increasing the fraction of misfolded protein present in the sample. Lastly, expressing at lower temperatures decreases the amount of protein degradation that occurs during expression. E. coli contain endogenous proteases that remain active during a standard 37℃ expression, which can lead to a loss of yield. While rare, autoproteolysis can also occur during 37℃ expression, an issue that is also abated by expressing at low temperatures. The addition of most commercially available protease inhibitors can be detrimental to cell growth, so lowering temperature is the best bet for preventing degradation events from occurring.
While there are many advantages to expressing at low temperatures, there are a number of downsides as well. First, low-temperature expressions decrease all metabolic processes associated with cell growth, including protein production. This can lead to a reduced yield, especially without compensating for this loss of activity by increasing the overall expression time. Second, for best results, specialized E. coli cell lines should be used. These cell lines typically have recombinantly altered chaperones or polymerases that are most active at lower temperatures. Lastly, low-temperature expressions greatly increase the time for sufficient quantities of protein to be produced, which will reduce experimental throughput. These downsides can be mitigated by expressing at room temperature, which will also serve to lessen the downsides of standard 37℃ expressions. Room temperature expressions require less time than cold expressions (though still not as fast as 37℃), have greater yield, and can be done using typical E. coli cell lines. The major takeaway from all of the factors should be that expression is not always ideal at 37℃, and the optimal temperature for expression should be optimized for each protein.
Which Temperature Should I Use?
Ideally, each protein should be fully characterized and its optimal expression temperature determined through repeated trials at each major temperature point (hot, cold, room temperature). Unfortunately, this process is laborious and incredibly time-consuming, so it is better to predict which temperature is best depending on the protein’s properties and working from there. A good place to start with a relatively unknown protein is the standard 37℃ protocol. If solubility is not an issue and the yield remains high, 37℃ should be used for the ideal temperature. If the protein is large, aggregation-prone, and/or is put into inclusion bodies, a low-temperature expression between 10℃ – 15℃ would be more suitable. For moderately insoluble proteins, expression conditions can be changed to room temperature to improve the overall yield and reduce time. While no substitute for a rigorous empirical comparison, these guidelines are a great place to start optimizing the temperature conditions for protein expression.