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.
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.
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
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 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.
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.
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.
For more detail, please see to Wikipedia.
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.
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 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%.
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 cell density fermentation VS Large scale fermentation（Picture from: Biologicscorp）
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.
Indirect feedback control
Direct feedback control
From: Lee SY, High cell-density culture of Escherichia coli [J], Trends in Biotechnology, 1996 March: 14(3):98-105.
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