Expression Technologies Inc.

Protein solubility

Factors determined by a chosen expression vector in a chosen system:

	Promoter strength: Under normal conditions, the weaker the promoter is, the higher protein solubility may be obtained. In addition, a weaker promoter gives higher yield for toxic proteins.
	Plasmid copy number: Low plasmid copy number plasmids normally give high amount of soluble proteins. These plasmids also give higher yield for toxic proteins.

Once an expression vector is chosen, all above factors are determined. Before constructing an expression vector, these factors must be taken into consideration.

Factors may be optimized in a standard laboratory:

Lower induction temperature

	Lower inducer concentration
	Try different host cell strains
	Express the target protein into different individual domains
	Delete the hydrophobic domain of the protein
	Use fusion partners (tags normally do not increase solubility significantly)
	Express in a different sub-cellular compartments or locations (cytoplasmic vs. secretory)
	Different expression systems (bacteria, yeast, insect, or mammal)
	Refolding: Denaturing chemicals, pH, salts, and detergents are important in protein refolding. Some proteins are easily refolded. Others cannot be refolded under a standard lab conditions.

Some of above factors can be easily optimized such as induction time, temperature, and inducer concentration. Others may require more molecular biology manipulation. These are all standard techniques and can be performed in most molecular biology labs.

Solubility can be improved by using our products:

Medium optimization:

	Trace metals, minerals, and vitamins may serve as prosthetic groups, co-factors, and ligands and increase solubility of recombinant proteins.
	pH may affect protein solubility. Phosphate and organic buffers may be used in the medium.
	Sugars may be used as inhibitors, carbon sources, and osmo-prectants and therefore affect protein solubility.

Protein co-expression:

	GroE, dnaKJ, peptidyl-proplyl isomerase (PPI), trigger factor (TF), thioredoxin
	heat shock proteins (hsp90α, hsp90β, hsp70, hsp60 and hsp56)
	DsbA, DsbB, DsbC and Protein disulfide isomerase (PDI)
	skp and secretion medium
	Natural partners

Proteins are soluble in their natural environments. A given natural environment provides all materials and tools necessary and sufficient for a particular protein expression. All above strategies are trying to provide all materials and tools needed for a recombinant protein expression. Different proteins require different materials and tools for its production. Therefore different tools are provided in the cell strains as co-expressed proteins. Our special media provide all known trace metals, minerals, and vitamins. In addition, we also include animal serum extracts which may contain some unknown trace metals, minerals and vitamins needed for some proteins. This is why some proteins are more soluble or completely soluble in our special media.

Multiple factors may be important for protein solubility. Combining our special medium, cell strains with molecular chaperones, and low protein synthesis rate (at low temperature up to 10 ⁰C), many proteins will become soluble and functional.

All these products can be easily implemented by switching media or simple transformation.

Protein solubility is a common problem in recombinant protein expression. Insoluble proteins appear to be packed in a special cellular compartment termed inclusion bodies. Many factors affect protein solubility. Medium nutrition, pH, osmotic pressure, ionic strength, rate of protein synthesis, amino acid composition, cellular location of expression, and cellular tools or chaperones are all important in protein solubility.

Medium nutrition is a critical factor in cell growth. In a regular medium such as LB, E.coli cells may reach OD₆₀₀ 2 to 3. In a richer medium with phosphate buffer such as TB, cells may reach OD₆₀₀ 5 to 8. When the medium is buffered with organic buffers such as sodium succinate/succinate acid and supplied with trace metals, minerals and vitamins and other nutrients, cells may reach OD₆₀₀ over 30. Trace metals, minerals and vitamins may not be needed for host cell growth, but they may serve as cofactor, prosthetic groups or ligands for the recombinant protein. Therefore they may be critical for protein solubility.

Strategies to improve protein solubility by medium optimization:

	Use special media containing trace metals, minerals and vitamins to medium. These chemicals may serve as cofactor, prosthetic groups or ligands which are essential for proper folding and protein solubility. In addition our special media are balanced with phosphate and organic buffers. They can support E.coli cells growth to OD₆₀₀ over 30.
	Include buffers in the medium to balance pH during cell growth. Low concentration of phosphate buffer may be exhausted at high cell density. High concentration of phosphate buffer is inhibitory to cell growth. Organic buffers such as citrate or succinate buffers may be used. These organic buffers can also serve as carbon source for cells. After cells use these organic acids as carbon source, the medium pH will increase in contrast with pH decrease when cells using sugars as carbon source.
	Supply sugars in the medium. Sugars increase osmotic pressure and lead to accumulation of so called osmo-protectants which stabilize the protein structure.

Strategies to improve protein solubility by reducing the rate of protein synthesis:

	Perform induction at lower temperature. This decreases the rate of protein synthesis and more soluble protein can be obtained.
	Use lower inducer concentration.
	Clone the cDNA into a low copy number plasmid.
	Express the protein under a relative weak promoter. T7 RNA polymerase has over 10 time higher rate of transcription than E.coli polymerase. T7 promoter can have higher protein yield, but high yield can also lead to protein insolubility.
	Combining our special media with lower temperature induction.

Some proteins are not soluble because of lacking cofactors or prosthetic groups. Without trace metals, minerals or vitamins, these proteins cannot be correctly folded or they become unstructured. Therefore they are insoluble. Others are not soluble because they are synthesized too fast to be correctly folded. Still other proteins are not soluble because they do not have sufficient tools or lack the tools to make them soluble. In this case, molecular tools or chaperones need to be expressed in the host cells.

Strategies to improve protein solubility by co-expressing molecular chaperones or tools:

	Co-express with E.coli GroE gene which encodes GroES and GroEL proteins.
	Co-express with DnaK and DnaJ proteins
	Co-express with peptidyl-proplyl isomerase (PPI). The peptide bond with proline has cis and trans conformations which cannot be changed unless the peptide bond is broken. Proteins with high proline contents may need PPI to be correctly folded. Trigger factor is shown to be the most efficient PPI in E.coli. Co-expression of proteins with trigger factor or other PPIs can significantly increase protein solubility and activity. Insolubility or loss of activity caused by incorrect proline conformation cannot be corrected by refolding.
	Co-express with thioredoxin (Trx). Trx has been shown to promote protein solubility if a protein is fused with it or co-expressed with it.
	Co-express with heat shock proteins (hsp). Hsps are shown to interact with hundreds of proteins. They are important in solubility and activity of many of these proteins.
	Co-express with protein promoting disulfide bond formation and isomerization such as DsbA, DsbB and DsbC. In this case, the protein needs to be expressed in periplasmic region of E.coli and therefore should contain a signal peptide. Commonly used signal peptides are from PelB, ompA and ompT. Periplasmic expression normally have lower yield than cytoplasmic expression. However it can often promote protein stability and reduce toxicity.
	Co-express with skp to increase the yield of periplasmic and secretory expression. Our special medium SecPro^TM Medium can increase protein yield hundreds of times. Up to tens or hundreds of milligrams of protein can be obtained from combination of our media and cell strains.
	Co-express with a protein natural partner. Some protein complex consists of subunits. Co-express all the subunits together can make the protein complex soluble and functional. Other proteins have natural partner in the biological system. Co-express them with their natural partners will increase their solubility and biological activity.

Protein co-expression may not be a very challenging task. When cell strains containing the molecular chaperones are available, the only experiment need to do is simple transformation and expression studies.

Strategies to improve protein solubility by fusion or by expressing as individual domain:

	Fuse with a polypeptide tag. A small polypeptide tag (< 30 amino acids) can only improve solubility of a small peptide. It will not have significant impact on solubility of protein larger than 10 kD.
	Fuse with a soluble protein such as thioredoxin, GST, MBP, or NusA. These fusion partners can increase solubility of many proteins.
	Delete hydrophobic or transmembrane domain can improve the solubility.
	Express the protein into different domains.

Examples:

	Increase protein yield, solubility, and activity in special media.
	Increase protein yield and stability in special media.
	Co-expression of nuclear receptor partners in a cell strain increases their solubility and biological activity.
	Co-expression of Smyd with hsp90 increases its yield, solubility and activity.

Related literatures of protein yield

Bacterial growth media
Protein yield
Protein toxicity
Plasmid DNA yield

Related products of protein yield

Bacterial E.coli growth media
DNA ladders or DNA markers
Expression vectors
Competent cells for cloning and expression
E.coli cell strains for protein expression

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