Cell-Free Systems As A Means Of Producing Medically Significant Recombinant Proteins
Introduction
There are 3 main methods of expressing and synthesizing protein, in vivo expression, chemical synthesis and the use of cell free systems (1). Cell free systems are techniques and methods that can be used to conduct recombinant protein synthesis outside of the cell, in vitro. Crude cell extracts are the basis of forming a cell free system. (2) Cell free systems use amin There are many advantages in using cell free-protein synthesis, also called CFPS, in comparison to in vivo techniques. CFPS produce higher yields of protein, faster procedure and do not rely on such capricious conditions in which the in vivo techniques are regulated by. By using cell- free techniques the expression of protein is more accessible to manipulate and alter, with the addition of chaperones or inducers such as IPTG. To achieve protein synthesis using cell free systems, involves a RNA polymerase solution along with a DNA template. (2) There are many cell free systems available today, the most cheapest is the uses of E. coli lysate, Rabbit reticulocyte, HeLa cell free system, Wheat Germ extracts, Yeast cell extracts, and insect cell extracts are also used.
Escherichia Coli Cell Extract the Main Microbial Cell-Free System
Cell lysate extracts are the basis for E. coli cell free systems when conducting protein synthesis. The most common species of bacteria to carry out cell free protein synthesis is Escherichia coli. E. coli is a gram negative bacteria. The bacteria is rod shaped. E. coli can change alter between aerobic respiration and anaerobic respiration which makes the species and facultative aerobe. The typical strains utilised in such techniques to produce medically significant recombinant protein are E. coli A19, D10 and BL21 (3). E. coli is the bacteria of choice as it produces high yields at a fast growth rate and it is a low cost system to express protein.
Preparation of E. Coli Cell Lysate
Cell lysates can be achieved by centrifuging to separate the soluble from the insoluble and then introducing lysis buffer to rupture the cell wall? In Haifa, Israel an experiment was conducted on the preparation of an E. coli lysate. A culture of BL21 (DE3) previously transformed with pAR1219 was grown and induced by Isopropyl-β-D-1- thiogalactopyranoside. After centrifuging, the insoluble was re-suspended in S30 lysate buffer. This buffer is the key step and is comprised of magnesium acetate, potassium acetate, tris acetate, 2-mercaptoethanol and dithiothreitol. The cells were then put into a high pressure homogeniser of 15,00psi. To separate the insoluble and soluble components of the bacteria a final centrifuge was conducted. The supernatant remaining is the E. coli lysate and can then be used to express protein without the use of whole, live cells.
Problems With E. Coli With Producing Recombinant Protein
As mentioned earlier, E. coli cell free protein expression is a fast technique that produces a relatively high yield of recombinant protein, however the quality of such protein is occasionally jeopardised. E. coli can be quiet sensitive and the addition of a plasmid can result in misfolded, aggregated protein. The use of chaperones such as GroEl and GrosEs is required to help fold the protein correctly. The larger oval shaped chaperone GroEl features a cavity which during hydrophobic interactions binds the foreign protein. GroEL and GroES work in tandem with GrosEs can bind to any end of the larger chaperone, these featuring resulting in the quintessential environment that allow for the folding of substrates. (5) One of the main reason not to use E. coli cell free systems is that E. coli components cannot carry out Post translational modifications while RRL can carry out limited Glycosylation and HeLa cell free systems can conduct Phosphorylation and Glycosylation modifications. Also with using prokaryotic protein synthesis substitutions of amino acids cannot be achieved. Saccharomyces cerevisiae is also used as a cell free system.
In particular, the mitochondria of the yeast cell. Yeast exhibits a unique property according to Scragg et al, the mictochondria of the fungus portrays a protein system analogous to that of bacteria in particular, E. coli. However, in the cytoplasm protein synthesis is executed in a comparable mechanism to mammalian cells, such as Chinese hamster ovarian cells (12). This property is based on the fact that Yeast are single celled microorganisms like bacteria but eukaryotic like mammalian cells. The extract used in yeast extract cell free protein synthesis can be prepared using the same basic techniques as E. coli lysate mentioned above. However, filtration of the smaller particles of the lysate is often demonstrated, usually in the form off column chromatography as utilised in a study in Colorado. The lysate must also undergo nuclease treatment before translation can begin. (14)In terms of the success rate of the production of recombinant protein using yeast extracts. The following results have been recordedHow e. coli components are actually used like what is lysate and how is it developed?More experiments and examples here.
Rabbit Reticulocyte Lysate
According to Ghadessy and Holliger, rabbit reticulocyte as a cell free protein synthesis system (RRL) has encountered many problems, the translation does not complete fully and discolouration of the RRL happened within minutes due to haemin oxidation. The study provides an effective solution for this CFPS to produce useable recombinant protein, with the use of a water in oil emulsion. (6) The yield of protein produced using RRL is relatively low in comparison to the high yield produced by E. coli cells and HeLa
Hela is a human cell line. They are some of oldest cells lines, over 60 years old. Hela cells are cancer cells and grow at rapid rates. The main features about these ancient cells is that they consistently divide and never die. They have been deemed as immortal cells. The human cell free system expresses protein relatively fast. In a study carried out in 2015, a transcription of mRNA was carried out using HeLa cell free system in under 75 minutes which is extremely quick for a transcription(7). E. coli is the main lysate used, however other options are available such as yeast cells, insect cells and wheat germ cells At Ehime University in Japan an experiment was conducted by removing α thionin and β thionin cysteine rich proteins from the wheat endosperm which are have been discovered to act as protein synthesis inhibitors. Tritin, an additional protein synthesis inhibitor, was also removed by extensive washing of the embryos (1). Recombinant proteins in the medical industry. Recombinant proteins are a vital component of the medical industry.
Antibodies, vaccines, drug delivery methods are comprised of these proteins. The reason recombinant proteins are so medically significant is due to the fact they can be manufactured at high volumes and relatively low cost. (8). CFPS are often chosen to express the desired proteins as previously mentioned can produce the Humunline, for example was the first pharmaceutical approved recombinant protein treatment for diabetes was cultivated by using E. coli cells. Over 151 European Medicine Aqgency (EMA) approved recombinant protein products have been approved. 45 of these products have been obtained from the application of E. coli which accounts for 29. 8% of all recombinant protein products developed (9). Work with recombinant proteins is still undergoing, in particular with vaccines for septic shock and HIV which currently is proving difficult in developing. Sullivan et al. explored the development of the monomeric gp120 vaccine for HIV. It is evident that the immunogens in the vaccine are unable to penetrate the wall of HIV-1 strains (10). A HIV vector called pNL4-3 was featured in a study in China to express the HIV protein in an Escherichia coli cell-free system using an E. coli DH5α strain lysate. Tricine-SDS PAGE analysis was carried out to screen for the protein. After expression the protein concentration of the chosen HIV protein, P10, was 85µg/ml. This level of protein is small but adequate for high throughput processes used in the pharmaceutical and medical industry. Fundamentally, E. coli as a cell free system for protein expression is essential to the prospects of producing an anti-HIV drug in the future. (11)Discussion: Round up about E. coli, maybe put in about the problems here and how they are overcome.
Conclusion
Cell free systems are extremely important for the pharmaceutical and medical industry as they are cheap, produce more yield and significantly faster than their in vivo counterparts. Based on the extracts properties and the properties of the protein they are expressing determines the suitability of the CFPS in developing certain vaccines and biopharmaceuticals. After analysing the various cell free systems that can be used for the production of medically significant recombinant protein, it is evident that Escherichia coli protein expression is the favoured technique. Although E. coli expression does not come without its own obstacles it is fast in vivo system that produces high yield of recombinant protein.
