How to Increase Soluble Protein Expression

Soluble Protein

How to improve protein solubility? To answer this question, we should know what factors contribute the protein solubility. For a particular protein, the electrostatic charge plays an important role in its solubility. For different proteins, the protein production processes also affect the solubility of particular protein.

The isoelectric points has the effects on protein solubility. Proteins are comprised of amino acids which interact with solution, like water. There is a technology known as isoelectric focusing, which can separate the proteins of protein mixtures. A mixture is placed in a polyacrylamide gel that has a pH gradient. An anode (positive electrode) and a cathode (negative electrode) are positioned at the low and high ends of the pH gradient, respectively. If a protein is located in the high pH region, it will be negatively charged and will move toward the anode. As the protein moves to a lower pH region, its surface charge will become less negative, and a pH region will be reached at which the protein net charge is zero (the isoelectric point). The protein will stop moving and, because different proteins have different isoelectric points, separation can be achieved.

The protein production processes also affect the solubility of particular protein. Simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (see to “A Simple Method for The growth medium Improving Protein Solubility and Long-Term Stability”).

From other researches, we can know the following factors also affects the protein solubility,

  • Factors which affect the rate of protein synthesis, like growth temperature, promoter type, plasmid copy ability, inducer concentration.
  • Growth medium.
  • Co-expressed chaperones and/or foldases
  • Cytoplasmic or periplasmic expression
  • Specific host strains
  • Fusion protein
  • Expression System

Now, let’s go to get soluble protein of interest.

Fusion Tags Enhance The Solubility Of Expressed Proteins

The soluble expression of heterologous proteins in E. coli remains a serious bottleneck in protein production. Although alteration of expression conditions can sometimes solve the problem, However, a systematic analysis of the utility of these solubility fusions has been difficult, and it appears that many proteins react differently to the presence of different solubility tags.

Today, there are a number of common solubility enhancing fusion tags that are used to express proteins in E. coli. In some cases, these tags double as affinity tags, not only facilitating soluble expression but also increasing the efficiency of protein purification. In other cases, these solubility tags have been combined with a simple His-tag,allowing the fusion partner to maintain its solublizing functionality and also double as an affinity tag. Additional affinity tags that can be combined with many of these solubility-enhancing tags are also available and have been successfully used to produce purified proteins.

 

Some commonly used solubility-enhancing fusion partners

More details from: https://www.researchgate.net/publication/7002912

New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli.

More details from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928792/