Overview of Protein Refolding

E.coli Protein Expression Service

Recombinant protein expression technology is widely used in protein functional research. Heterologous gene expression commonly leads to production of inclusion bodies. To obtain souble protein, inclusion bodies must be refolded into an active form through refolding methods. Refolding is initiated by reducing the concentration of denaturant to solubilize inclusion bodies.

Protein Refolding

What is the inclusion body?

Inclusion bodies(IBs) are the formation of insoluble protein particles which are caused by expression of heterologous genes, especially in highly efficient expression in Escherichia coli cells. The protein with biological activity in cells are usually soluble. IBs are aggregates of unfolded and with no biological activity. Therefore, IBs must be dissolved by denaturing agent and refolding methods.

Formation of inclusion body

1) Over expression is more likely to cause inclusion bodies formation
2) The amino acid composition of recombinant proteins: the more with sulfur amino acid, the easy to form
3) The expression environment of recombinant protein: over high fermentation temperature
4) In eukaryotes, the enzymes of post-translational modification are lacked, which results in the accumulation of intermediates. So IBs are easy to form in cells

Refolding Methods

    • Dialysis, dilution and ultrafiltration – These three methods are traditional and generally used in the protein refolding. The recovery rate is very low and it is difficult to be separated from hybrid proteins. Dilution method is time-consuming and easy to form aggregates of inactive proteins, not suitable for industrial production.
    • Gel filtration chromatography – Gel filtration chromatography is used for refolding high concentration of protein. The recovery rate is higher than dilution method and this method can also be applied to purification of protein.
    • Add accelerant – The function of accelerant can stabilize the natural structure of folding protein, increase the solubility of refolding intermediate or increase the unfolded protein solubility.
    • LC (liquid chromatography) – Liquid chromatography is one of the effective methods for purifying proteins, which becomes an essential tool for recombinant protein purification, like hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC), gel exclusion chromatography (SEC), affinity chromatography (AFC), etc. Compared with the traditional dilution and dialysis method, liquid chromatography has the below advantages:
      1) Denaturing agent can be quickly removed.
      2) The adsorption of denatured protein are obviously reduced, therefore to avoid the generation of precipitation.
      3) It can separate the protein of interest from hybrid proteins, and make renaturation and purification at the same time.

How to validate refolding

1) Gel electrophoresis
2) Spectroscopic method – It only be used for refolding process in research. Ultraviolet difference spectra , Fluorescence spectra and Circular Dichroic Spectroscopy are included.
3) Chromatographic process – IEX, RP-HPLC, CE, etc
4) Immunological methods – Elisa, Western blot

Chemical protein synthesis definition

Chemical protein synthesis is named in contrast to the biological protein synthesis. The synthesis is by coupling the carboxyl group or C-terminus of one amino acid to the amino group or N-terminus of another. It is also called peptide synthesis, because the length of synthesized amino acid sequence is limited by current synthesis technology. The most length of synthesized is about 50 AAs.

Peptide Synthesis
Peptide Synthesis

Protein synthesis is the nature biological process with two big steps, transcription and translation. The transcription is from DNA to mRNA and the translation is from mRNA to protein. For more details, please read more.

Chemical protein synthesis

The chemical total synthesis of proteins, even of small proteins, is by no means trivial and a tedious enterprise. Solid phase peptide synthesis is the most powerful method for the synthesis of small- to medium-sized peptides (5-50 amino acids). The iterative coupling steps accumulate by-products, the separation of which is difficult with longer sequences. Convergent methods avoid the “cumulative disaster” of linear synthesis. Access to large peptides can be provided by employing medium-sized peptide segments that are easily available by solid phase synthesis. The most successful approach involves the segment coupling of unprotected peptide segments.
The bottleneck of native chemical ligation is the tedious peptide thioester synthesis. Moreover cysteine is rare in protein sequences and in many cases an artificial cysteine residue has to be introduced for the sake of ligation.

Protein production

For more detail, please see to Wikipedia.

Protein synthesis vs protein production

Protein synthesis is the nature biological process with two big steps, transcription and translation. The transcription is from DNA to mRNA and the translation is from mRNA to protein.

The protein production is not a totally nature biological process, which integrates recombinant DNA technology and the nature biological protein synthesis process. There are four types of preferred protein expression systems, including E.coli, yeast, insect and mammanlian system. It aims to improve the expression level and the solubility of protein.

Biological Protein Synthesis
Gene Transcription, Translation, and Protein Synthesis.
(© 2001 Terese Winslow)
See to: http://stemcells.nih.gov/info/2001report/pages/appendixA.aspx

Protein synthesis

The making of the various types of protein is one of the most important events for a cell because protein not only forms structural components of the cell, it also composes the enzymes that catalyze the production of the remaining organic biomolecules necessary for life.

The DNA housed in the nucleus is too large to move through the nuclear membrane, so it must be copied by the smaller, single-stranded RNA (transcription), which moves out of the nucleus to ribosomes located in the cytoplasm and rough endoplasmic reticulum to direct the assembly of protein (translation). The genes do not actually make the protein, but they provide the blueprint in the form of RNA, which directs the protein synthesis.


Transcription occurs in the cell nucleus and represents the transfer of the genetic code from DNA to a complementary RNA. The enzyme RNA polymerase Attaches to and unzips the DNA molecule to become two separate strands. Binds to promoter segments of DNA that indicate the beginning of the single strand of DNA to be copied. Moves along the DNA and matches the DNA nucleotides with a complementary RNA nucleotide to create a new RNA molecule that is patterned after the DNA. The copying of the DNA continues until the RNA polymerase reaches a termination signal, which is a specific set of nucleotides that mark the end of the gene to be copied and also signals the disconnecting of the DNA with the newly minted RNA.


Translation is the conversion of information contained in a sequence of mRNA nucleotides into a sequence of amino acids that bond together to create a protein. The mRNA moves to the ribosomes and is read by tRNA, which analyzes sections of three adjoining nucleotide sequences, called codons, on the mRNA and brings the corresponding amino acid for assembly into the growing polypeptide chain. The three nucleotides in a codon are specific for a particular amino acid. Therefore, each codon signals for the inclusion of a specific amino acid, which combines in the correct sequence to create the specific protein that the DNA coded for.
The assembly of the polypeptide begins when a ribosome attaches to a start codon located on the mRNA. Then tRNA carries the amino acid to the ribosomes, which are made of rRNA and protein and have three bonding sites to promote the synthesis. The first site orients the mRNA so the codons are accessible to the tRNA, which occupy the remaining two sites as they deposit their amino acids and then release from the mRNA to search for more amino acids. Translation continues until the ribosome recognizes a codon that signals the end of the amino acid sequence. The polypeptide, when completed, is in its primary structure. It is then released from the ribosome to begin contortions to configure into the final form to begin its function.

Protein production

Protein production is the expression of proteins that have been produced by recombinant DNA techniques. This process enables these substances to be made in large quantities. Such mass production is done both for laboratory study and for industrial production.
Proteins are chains of amino acids, encoded by DNA. The genes that code for these proteins are put into special vectors, or units of DNA. Vectors are chosen that will produce large amounts of the desired protein. This is known as overexpression.
Overexpression is done in special host cells. Sometimes the hosts are bacteria or yeast. In cases where the proteins are from mammals, the hosts are frequently insect or mammalian cell lines. A large number of kits are commercially available to facilitate both the cloning of the gene, and the subsequent recombinant protein production. In order to over-express the protein, the gene sequence should be optimized to adapter the expression host and the expression condition should also be optimized. Especially, the e.coli system is the most of used system, because its success rate is the highest, more than 98%.

Transient Transfection Introduction


Transient transfection is a well-established method to rapidly express recombinant proteins from mammalian expression system. Transient transfections can be reliably and readily scaled up to handle milliliters to tens of liters of cells in suspension culture and obtain milligrams to grams of recombinant protein in a process that requires only days to weeks. Transient gene expression in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells.

Transient transfection introduction from: https://www.researchgate.net/publication/

For some applications of transient transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded. Cell lines expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution.

Transient transfection introduction from: https://en.wikipedia.org/wiki/Transfection#Stable_and_transient_transfection

High Cell Density Fermentation – A Efficient Fed-Batch Fermentation Technology

High Cell Density Fermentation VS Stand Large Scale Fermentation

High cell density fermentation by fed-batch strategies is one of the most cost-effective means of achieving high yields for the production of large scale recombinant proteins in the bio-industry. In fed-batch cultures, cell mass and productivity are maximized by adjusting culture conditions, including temperature and pH, the composition of the feed media, and the substrate feed rate. As one of the most critical factors to the success of high cell density culture, various nutrient feeding strategies have been developed.

High Density Fed-batch Fermentation VS Stand Fermentation

High cell density fermentation VS Large scale fermentationPicture from: Biologicscorp

Feeding methods in fed-batch culture

Without feedback control

  • Constant feeding – feeding nutrient at a predetermined (constant) rate. As a result, the specific growth ratecontinuously decreases.
  • Increased feeding – feeding nutrient at an increasing (gradual, stepwise, or linear) rate. The decrease in specific growth rate can be compensated within a range.
  • Exponential feeding – feeding nutrient at an exponential rate to achieve constant specific growth rate.


Specific growth rate is maintained at a constant level by exponential feeding in most cases, but may deviate when unexpected conditions arise during culture. When it occurs, feedback control is required in the process to achieve constant specific growth rate.

With feedback control

Indirect feedback control

  • DO-stat – feeding nutrient when there is a rise in the concentration of dissolved oxygen (DO), which results from depletion of the substrate.
  • pH-stat – feeding nutrient when the pH rises after depletion of the principal carbon source.
  • Carbon dioxide evolution rate (CER) – the most frequently method to maintain the specific growth rate. The CER is roughly proportional to the rate of consumption of the carbon source using a mass spectrometer.
  • Cell concentration – the nutrient feeding rate determined from the cell concentration is measured by a laser turbidimeter.

Direct feedback control

  • Substrate concentration control – nutrient feeding is directly controlled by the concentration of the principal carbon source (e.g. an on-line glucose analyzer in the fermentor).

From: Lee SY, High cell-density culture of Escherichia coli [J], Trends in Biotechnology, 1996 March: 14(3):98-105.

Fermentation Definiton

Industrial fermentation is the intentional use of fermentation by microorganisms such as bacteria and fungi to make products useful to humans. Fermented products have applications as food as well as in general industry. The rate of fermentation depends on the concentration of microorganisms, cells, cellular components, and enzymes as well as temperature, pH and for aerobic fermentation oxygen. Product recovery frequently involves the concentration of the dilute solution. Nearly all commercially produced enzymes, such as lipase, invertase and rennet, are made by fermentation with genetically modified microbes. In some cases, production of biomass itself is the objective, as in the case of baker’s yeast and lactic acid bacteria starter cultures for cheesemaking. In general, fermentations can be divided into four types:

1. Production of biomass (viable cellular material)

2. Production of extracellular metabolites (chemical compounds)

3. Production of intracellular components (enzymes and other proteins)

4. Transformation of substrate (in which the transformed substrate is itself the product)


These types are not necessarily disjoint from each other, but provide a framework for understanding the differences in approach. The organisms used may be bacteria, yeasts, molds, animal cells, or plant cells. Special considerations are required for the specific organisms used in the fermentation, such as the dissolved oxygen level, nutrient levels, and temperature.

Information from: wikipedia.org/wiki/Industrial_fermentation#Production_of_biomass

Soluble Protein Test Introduction and Method

Introduction of soluble protein test

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/

soluble protein introduction
Picture from: Soluble expression of recombinant proteins in the cytoplasm of
Escherichia coli

Test Method For Solubility Determination:

soluble protein service

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.

BiologicsCorp proudly launches this soluble protein expression service – another first in the industry. We have tested many variables, including codon optimization, strain, charperones and foldases, and others. Give SupernateIN™ a try, especially if you prefer the protein expression in soluble form. We are confident and can guarantee success – no soluble fraction for target protein, no payment for expression service. Learn more

Recombinant Protein Articles


Protein expression in e.coil

Production of Recombinant Proteins – Challenges and Solutions (PDF)
Abstract: Efficient strategies for the production of recombinant proteins are gaining increasing importance,as more applications that require high amounts of high-quality proteins reach the market. Higher production efficiencies and, consequently, lower costs of the final product are needed for obtaining a commercially viable process. In this chapter, common problems in recombinant protein production are reviewed and strategies for their solution are discussed. Such strategies include molecular biology techniques, as well as manipulation of the culture environment.

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications
Abstract: Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology.

So you Need a Protein – A Guide to the Production of Recombinant Proteins
Abstract: The field of biotechnology owes a great deal to the ability to produce recombinant proteins, which can be made
in far greater abundance than many native proteins, and are more easily quality controlled. There is a great need for individual
proteins to be produced for research purposes. This review is aimed at researchers who are not experienced at protein
expression, but find that they have a need to produce a recombinant protein. We detail the major expression systems
that will be commonly used in the laboratory situation- bacterial, yeast and insect cell culture. The application of each,
and the relative advantages/disadvantages are discussed.

History of Recombinant Protein

Herb BoyerWhat is the History of Recombinant protein? The method of recombinant DNA was initially planned by a graduate student, Peter Lobban, along with a biochemist, A. Dale Kaiser at the Stanford University.

During then years,1972–74, the method was then acknowledged by Stanley Norman Cohen, an American geneticist Chang, Herbert Boyer, a addressee of the 1990 National Medal of Science. In 1973, they published their predictions in journal “Enzymatic end-to-end joining of DNA molecules” which explained the methods to separate and intensify genes or DNA segments and introduce them into an additional cell with accuracy.

In 1977, an advance in the field of recombinant DNA technology took place when Herbert Boyer created the biosynthetic “human” insulin, a group of biosynthetic human insulin products.

Bacterial recombinant protein expression service       Mammalian recombinant protein expression service

Recombinant Protein Definition

Recombinant protein is a manipulated form of protein, which is generated in various ways to produce large quantities of proteins, modify gene sequences and manufacture useful commercial products. The formation of recombinant protein is carried out in specialized vehicles known as vectors. Recombinant technology is the process involved in the formation of recombinant protein.
— recombinant protein definition from http://www.ehow.com/about_5407160_recombinant-protein-definition.html

Recombinant Protein is a protein encoded by a gene — recombinant DNA — that has been cloned in a system that supports expression of the gene and translation of messenger RNA (see expression system). Modification of the gene by recombinant DNA technology can lead to expression of a mutant protein. Proteins coexpressed in bacteria will not possess post-translational modifications, e.g. phosphorylation or glycosylation; eukaryotic expression systems are needed for this.
— recombinant protein definition from http://www.answers.com/topic/recombinant-protein

Recombinant DNA (rDNA) molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure; they differ only in the sequence of nucleotides within that identical overall structure. Consequently, when DNA from a foreign source is linked to host sequences that can drive DNA replication and then introduced into a host organism, the foreign DNA is replicated along with the host DNA.
Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein will not necessarily be produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence.
— recombinant protein definition from https://en.wikipedia.org/wiki/Recombinant_protein