StudentShare
Contact Us
Sign In / Sign Up for FREE
Search
Go to advanced search...
Free

Downstream Processing of Recombinant Proteins in E.coli - Essay Example

Cite this document
Summary
This essay declares that escherichia coli is the most widely used system for the production of recombinant proteins. The main advantage of using E.coli is the highly characterized DNA and the growth rate. E.coli can be grown under laboratory conditions in the Luria Bertani Broth…
Download full paper File format: .doc, available for editing
GRAB THE BEST PAPER93.4% of users find it useful
Downstream Processing of Recombinant Proteins in E.coli
Read Text Preview

Extract of sample "Downstream Processing of Recombinant Proteins in E.coli"

 Escherichia coli is the most widely used system for the production of recombinant proteins. The main advantage of using E.coli is the highly characterized DNA and the growth rate. E.coli can be grown under laboratory conditions in the Luria Bertani Broth and the genetic manipulation is easy. The heterologous proteins are produced in E.coli as insoluble bodies, and are called as inclusion bodies. (Singh and Panda 2005). The inclusion bodies contain densely packed denatured protein molecules. These protein molecules are then refolded and brought back to active form. Hence for the high recovery of the active protein molecule, solubilization and refolding parts must be of high precision. Inclusion bodies consist of polypeptides of the recombinant protein. They are the inactive secondary –like structures. Thus, the isolation and purification of the protein are simple. The activity of the unfolded protein can be brought out by using mild solubilizing conditions. This will help in the high recovery of the bioactive protein than that compared to solubility using high chaotropic agent. The following Downstream processing steps can be used for the production of the recombinant protein from the inclusion bodies Fermentation Cell disruption Centrifugation Solubilization Refold Chromatographic purification Fermentation: Overnight culture centrifuge pellet suspension in sodium phosphate buffer cell lysis Centrifugation. The recombinant E.coli is grown in LB medium with antibiotics such as kanamycin or ampicillin or chloramphenicol based on the plasmid. The flasks are shaken at rpm around 150 -250 and the temperature is maintained at 37 degree Celsius. After the cells have reached the log phase, IPTG is added and further shaken for 4-6 hours. The cells are centrifuged and re-suspended in the 50 mM sodium phosphate buffer. The cells are lysed using the homogenizer or sonicator. The cell suspension is centrifuged and filtered using 0.45 µm polyethersulfonate membrane. Solubilization: Many protein-specific methods are available for the increased solubility of the recombinant proteins in E.coli. To recover the soluble proteins, strong denaturants like urea, guanidinium hydrochloride are used. The solubilization is carried out under reducing conditions. These inclusion bodies must be washed well before solubilization. Solubilizing agents such as thioredoxin are known to improve the solubility of the proteins. Gene fusion techniques are equally good for the separation of the proteins. Maltose binding protein and Glutathione-S- transferase are found to bind well with the protein and can be removed by using the affinity chromatography techniques. (Ghosh et al. 2004). Refolding: Refolding is performed using dilution or diafliltration in buffers of low concentration chaotropic agents. The removal of the denaturant will favor the refolding process. The specific folding conditions vary between the proteins. Chromatographic purification: Arginine Sepharose Chromatorgraphy is a type of Affinity chromatography column where L-arginine is immobilized on Sepharose 4B using epoxy coupling method. It has a hydrophilic spacer, alkylamide bond and stable ether. L-arginine is linked to Sepharose through the alpha – amino group and the sample substances are free to interact with the alpha carboxyl groups in the chromatography. It is highly used for the separation of many serine proteases from a wide range of organisms. (Gelifesciences.com). Stably Folded proteins : Stably folded recombinant protein are of major interest for the scientists. The recombinant proteins are well expressed in E.coli. E.coli can be used to produce many human proteins, protein complexes, bacterial proteins and membrane proteins of human at higher expression levels than other bacteria. The probability of expression of proteins decreases with increase in molecular weight above 60 kDa. For high expression profiles, the vectors with kanamycin or chlormaphenicol resistance can be used. (Graslund et al. 2008). The Recombinant E.coli can be grown in the LB medium with one of the antibiotic kanamycin or ampicillin or chlormaphenicol, based on the plasmid used. E.coli is grown with gentle agitation and at room temperature till the log phase and then IPTG is added to it and further left for 4 – 6 hours. Extraction of the enzyme from the cell can be carried out by using mild methods such as cell lysis by osmotic shock, Enzymatic digestion of the cell wall and to extract the intracellular proteins and moderate method such as Grinding the cells with some abrasive like sand. Other methods used for cell lysis are Ultrasonication, bead milling, homogenizer, French press and Fractional precipitation. (Ahmed 2004). After the lysis, the protein is precipitated using Ammonium sulphate or polyethylene glycol or acetone. The widely used technique for the precipitation of the protein is Ammonium sulphate precipitation. After precipitation, dialysis is performed to remove the salt content and small molecules present along with the protein. After dialysis, suitable chromatography column is used for the purification and quantification of the protein. Since the enzyme has a greater affinity for L-Arginine - Sepharose media, Sepharose 4D affinity chromatography column is prepared and the enzyme is purified. During affinity chromatography, the parameters such as sample concentration, pH, buffer composition, flow rate and temperature are optimized for optimal purification. The pH of the sample should always be the same as the binding buffer. After the binding, the recombinant molecule is eluted using specific elutant such as 2M of NaCl, urea or guanidine hydrochloride. If the proteins are absorbed strongly to the column, then 6 M guanidine hydrochloride or 8 M Urea is used. (Gelifesciences.com). Fermentation ( LB broth till log phase) Cell lysis ( homogenizer or Ultrasonication or Fractional precipitation) Removal of cell pellet and Ammonium sulphate salt precipitation Salting out by Dailysis ( to remove the small molecules and the salt from the enzyme solution) L- Arginine – Sepharose Affinity Chromatography Eluting out using 2M Sodium chloride or Urea or Guanidium hydrochloride. Thus the 14 kDa recombinant protein can be produced inside the E.coli and extracted from E.coli using the standard techniques. The folded protein and the insoluble aggregates thus extracted and purified, is verified by performing Polyacrylamide Gel Electrophoresis with molecular marker. Since the enzyme has a disulfide bond, the ability to bind with the Arginine Sepharose column is high and the elution process is simple. The similarities between two techniques are the fermentation, cell lysis techniques. The centrifugation will vary as it lies in two forms folded protein and insoluble aggregate. The process of obtaining the protein in the form of folded protein is very advantageous. The insoluble aggregates require some additional steps to obtain an active protein. References: Ahmed, H., 2004. Principles and Reactions of Protein Extraction, Purification, and Characterization, CRC Press. Structural Genomics Consortium, Gräslund, S., Nordlund, P., Weigelt, J., Hallberg, B. M., Bray, J., Gileadi, O., Knapp, S., et al., 2008. Protein Production and Purification, National Methods, Vol.5, No.2, pp. 135- 46. Gelifesciences. Arginine Sepharose. Available from: http://www.gelifesciences.co.jp/tech_support/manual/pdf/71710100ac.pdf (Accessed on April 6, 2014). Ghosh, S, Rasheedi, S, Rahim, SS, Banerjee, S, Choudhary, RK, Chakhaiyar, P, Ethesham, NZ, Mukhopadhyay, S and Hasnain, SE., 2004. Method for Enhancing Solubility of the expressed Recombinant Proteins in Escherichia Coli, Biotechniques, Vol.37,No.3,pp. 418 -420. Singh, SM and Panda, AK., 2005. Solubilization and Refolding of Bacterial Inclusion Body proteins, Journal of Bioscience and bioengineering, Vol.99, No.4, pp. 303 - 310. Read More
Cite this document
  • APA
  • MLA
  • CHICAGO
(“Downstream Processing of Recombinant Proteins in E.coli Essay”, n.d.)
Retrieved from https://studentshare.org/biology/1637252-downstream-processing
(Downstream Processing of Recombinant Proteins in E.Coli Essay)
https://studentshare.org/biology/1637252-downstream-processing.
“Downstream Processing of Recombinant Proteins in E.Coli Essay”, n.d. https://studentshare.org/biology/1637252-downstream-processing.
  • Cited: 0 times

CHECK THESE SAMPLES OF Downstream Processing of Recombinant Proteins in E.coli

Emulsifying and Emulsion Stabilizing Role of Proteins

Different proteins in a food system exhibit different degree of stabilizing and emulsifying properties due to their variation in their molecular structure, functionality, flexibility, hydrophobicity, charge, and molecular size.... Food emulsions are oil-water dispersion systems, in which proteins play the emulsifying and emulsion stabilizing role.... After adsorption on the interface, molecules exhibit a conformation that is influenced by molecular flexibility and environmental factors, including ionic strength, presence of low molecular weight emulsifiers or other proteins, and pH....
6 Pages (1500 words) Essay

Montage Is the Art and Technique of Motion-Picture Editing

recombinant Art with relation to montages and tribute videos Introduction: According to the Columbia Encyclopedia montage is the art and technique of motion-picture editing in which contrasting shots or sequences are used to affect emotional or intellectual responses.... rom oulipo to recombinant poetics Interaction with different forms of generative production enables one to dynamically explore emergent meaning.... There is an interesting commonality to generative literary, artistic, and musical production that is relevant to the OULIPO, recombinant Poetics, as well as techno-audio remix culture....
1 Pages (250 words) Essay

The Uses of Recombinant DNA Technology in Medicine

By cloning their genes via PCR amplification and cloning into bacterial expression hosts, we can circumvent these issues and skip past the rate-limiting step of purification since cloning produces proteins in bulk.... The paper “The Uses of recombinant DNA Technology in Medicine” evaluates the engineering of DNA, into a purely synthetic combination that would not usually be found in nature.... Insulin, therefore, has become far more available for treatment with the advent of recombinant DNA technology....
3 Pages (750 words) Assignment

Cellular processing of Alzheimers amyloid precursor protein

Cellular processing of Alzheimer's amyloid precursor protein (APP) is carried out at multiple levels through a set of enzymes, secretases.... Cellular processing of Alzheimer's amyloid precursor protein This transport has been found to be Tyr653 dependent within cytoplasmic tail; what needs to be explored is cytoplasmic factors involved in the transport of APP.... Apart from processing, there is vesicular trafficking which result in the movement of proteins across sub-cellular compartments....
3 Pages (750 words) Essay

The Positive Effects of Enzymes

The… Once we understand the efficacy and effectiveness of these enzymes we will have a newfound alternative to maintaining our health and preventing proteins: Scientists have identified several factors that help lengthen the span of natural human lives.... The objective of the book being analyzed is to help the reader appreciate the positive effects of certain essential proteins called Enzymes.... Many refer to enzymes as special proteins that are biologically active or contain energy....
2 Pages (500 words) Essay

DNA Sequence Analysis, Primer Design, Protein Expression, and Mutagenesis Assessment

coli where crystallization formation can be essential for post mutation testing.... RecA is a recombinant gene and when in the presence of single-stranded DNA, can behave as a catalyst in the hydrolysis process of ATP.... The paper states that when lysine is removed from the genetically engineered genome of an organism, the organism becomes dependant on synthetic forms of lysine....
3 Pages (750 words) Assignment

Production of tpa using eukaryotic n prokaryotic cells

From the case study, it is evident that production of recombinant tPA in CHO (mammalian) cells is less costly than in e.... tPA produced in e.... Since the proteins in the bacteria are produced in low concentrations (about 2.... Also, these cells are usually susceptible to contaminations resulting from microbial infections. From the case study, it is evident that production of recombinant tPA in The Production Of tPA using eukaryotic n prokaryotic cells The Production Of tPA using eukaryotic n prokaryotic cells Cell culture is the growing of prokaryotic or eukaryotic cells under controlled conditions....
2 Pages (500 words) Essay

GFP Green fluorescent protein

Transgenic organisms of various species such as e coli, C elegans, Drosophila were made to glow by inserting GFP reporter gene.... In the process, one reporter acts as an internal control and other responder to analyze, such as incorporation of GFP and YFP in e.... coli, making construct as recA'::egfp and grpE'::dsRedExpress.... Reporter genes are tagged with multicoloured fluorescent proteins which aid in identifying the chemical effects prevailing in the environment....
2 Pages (500 words) Essay
sponsored ads
We use cookies to create the best experience for you. Keep on browsing if you are OK with that, or find out how to manage cookies.
Contact Us