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Endotoxin Removal by Triton X-114 partitioning

Published on September 11, 2012 by in Home

A variety of methods have been developed in order to remove endotoxins from protein solutions, with one of the more popular being separation by phase using non-ionic detergent Triton X-114. Triton X-114 detergent is homogeneous at cold temperatures, but once above a critical temperature of 22°C, its solution separates into an aqueous phase, where hydrophilic proteins are found, and a detergent phase, where impurities such as endotoxins and other hydrophobic molecules are captured. Multiple cycles of cooling-heating-phase separation have to be repeated until the remaining endotoxin is below the threshold limit.  It should be noted that this method cannot be used if a target protein happens to be hydrophobic and partitions into a detergent-enriched phase.

 

Endotoxin Removal

Endotoxin Removal by Triton X-114 partitioning

 

This is a simple and cheap procedure.   In most cases the repeated cooling heating process has proven to remove endotoxins without affecting any significant losses in protein yields and biological activities.  However, it has to be noted that residual Triton will remain in the sample at <0.02% concentration and it cannot be dialyzed out due to its large micelle size.

 

 

Lopes, Andre M., Magalhaes, Perola O., Mazzola, Priscilla G., Penna, Thereza C. V., Pessoa, Adalberto Jr., and Rangel-Yagui, Carlota.  “Methods of Endotoxin Removal from Biological Preparations: a Review.” J Pharm Pharmaceut Sci. 2007, 10(3):388-404.

 

 
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Endotoxin Removal: General Information

Published on August 20, 2012 by in Home

Endotoxins are toxic protoplasms that are released when bacteria grow, die, and disintegrate. These endotoxins are lipopolysaccharides, which reside in the outer membrane of gram-negative bacteria. The gram-negative cell wall is composed of a thin, inner layer of peptidoglycan and an outer membrane of phospholipids, lipopolysaccharides, and lipoprotein.

 

PROPERTY              ENDOTOXIN
Chemical nature Lipopolysaccharide
Relationship to cell Outer membrane
Size 10 kilodalton
Isoelectric point 2.5
Denatured by boiling No
Antigenic Yes
Form toxoid No
Potency >100 micrograms
Specificity Low degree
Enzymatic activity No
Pyrogenicity Yes

 

The lipopolysaccharides, or endotoxins, are made up of O-antigens, a core polysaccharide, and lipid A, which is made up of glucosamine and fatty acids and is responsible for the toxic effects. The toxins are continuously shed from the outer membrane of bacteria, and released into the environment. When proteins are purified from E. coli, the unwanted endotoxin by-products must be removed from the final product for manufacturing processes and in vivo applications.

 

lipopolysaccharide structure

 

The toxicity of endotoxins makes their removal an important step for protein application in several biological assays and for safe release of product, as required by the FDA. The endotoxin limit for injections can be calculated by dividing the minimum pyrogenic dose of endotoxin per kilogram by the maximum dose of product per kilogram per hour.

 

TYPE           INTRATHECAL ENDOTOXIN LIMIT
Injectable drug          Yes .2 endotoxin unit per kilogram
Injectable drug           No 5 endotoxin units per kilogram
Medical device          Yes .06 endotoxin units per milliliter
Medical device           No .5 endotoxin units per milliliter
Radiopharmaceuticals          Yes 14 endotoxin units per volume
Radiopharmaceuticals           No 175 endotoxin units per volume
Sterile water            – .25 endotoxin units per millimeter
Assay            – Positive product controls recovery >50%

 

 

 

 

 

 

 

 

 

 

 

 

Check out http://www.qimedical.com/links/Endotoxin_Limits_Document_2011.pdf to compare various endotoxin limits for common injectables set by the United States Pharmacopoeia

 

Increases in the production of cytokine, prostaglandin, acid phosphatase, fibrinolytic inhibitor, collagenase production, nerve growth factor, factor B, polypeptides, platelet activating factor, adhesion inhibitor, adhesion molecule-1 and procoagulant can result from endotoxin consumption, and thus can cause multiple responses which vary between animals and humans.

 

ANIMALS    HUMANS
Fever Fever
Decrease in blood pressure Decrease in blood pressure
Disseminated intravascular coagulation Intravascular coagulation
Hypotension Hypotension
Shock Shock
Change in instincts for survival Inflammation
Severe blood flow changes Septic shock
Changes in metabolic hormones Tissue injury
Poor growth Vasodilation
Changes in white blood cell counts Fibrinolysis
Death Hemorrhaging

 

Normally, when animals are injected with even small doses of endotoxin, most experience a latent period, physiological distress such as diarrhea, prostration, and shock, and then finally death. They vary in their susceptibility to endotoxin. With humans, it’s more complex. The endotoxins bind to a lipid binding protein in the serum and are transferred to the CD14 receptor on the cell membrane. It is then transferred to the MD2 protein, which is associated with the TLR$, which triggers a cascade of signals for cells to secrete pro-inflammatory molecules, which can lead to various responses.

Endotoxin

Endotoxins are a natural part of gram-negative bacteria such as Acinetobacter, Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria, Enterobacter, Erwinia, Escherichia coli, Franciscella, Helicobacter, Hemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio, and Yersinia.  Any samples that are derived from gram-negative bacterial cultures have a high presence of endotoxins.  In addition, adventitious endotoxins can be found everywhere bacteria had have a minute presence including dust, dirt, and air.  In laboratory environment endotoxins can come from water, sera, media, reagents, buffers, plasticware and glassware.  Since endotoxins have high stability in both high temperature and high pH, they cannot be removed by sterilization.  Due to its wide-spread presence and low thresholds for pyrogenic reaction, it is essential to remove endotoxins from samples that are destined for in vivo use, from therapeutics to ensure the health of the consumers.

Endotoxin removal methods for protein solutions are usually based on affinity, ion-exchange chromatographies, or detergent phase partitioning.  Multiple cycles must be done to completely remove endotoxins and make products non-pyrogenic. (www.arvysproteins.com).

 

 

 

 

  • Affinity Chromatography: Principles and Methods, AC ed.; Amersham Pharmacia  Biotech: Upsala, Sweden. 2001.
  • Bordier C. “Phase separation of integral membrane proteins in Triton X-114 solution.”   The Journal of Biological Chemistry. Feb 1981, 256:1604-1607.
  • Cohen J and Davis B. “Endotoxin Removal Devices for the Treatment of Sepsis and  Septic Shock.” The Lancet Infectious Disease. 2011 Jan;11(1):65-71.
  • Guadagni G, Perego A, Shoji H, and Tani T. “Extracorporeal Removal of Endotoxin: The Polymyxin B-immobilized Fiber Cartridge.” Contributions to Nephrology. 2010;167:35-44.
  • Huang Y, Li Y, Luo J, Ma G, Su Z, Xing H, and Zhang L. “Effect of Affinity Medium and Solution Conditions on Endotoxin Removal from Protein Solutions.” Chinese Journal of Biotechnology. 2010 Nov;26(11):1584-95.
  • Lopes, Andre M., Magalhaes, Perola O., Mazzola, Priscilla G., Penna, Thereza C. V., Pessoa, Adalberto Jr., and Rangel-Yagui, Carlota.  “Methods of Endotoxin  Removal from Biological Preparations: a Review.” J Pharm Pharmaceut Sci. 2007, 10(3):388-404.
  • Ohno N, Morrison DC. “Lipopolysaccharide Interaction with Lysozyme: Binding of Lipopolysaccharide to Lysozyme and Inhibition of Lysozyme Enzymatic Activity.” J Biol Chem 264:4434–4441. 1989.
  • Ryan, John. “Endotoxins and Cell Culture: Technical Bulletin.” Corning Incorporated: Corning, NY.  2008. 25 June 2012.             http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/cc_endotoxins_tc_305_rev1.pdf.
  • Todar, Kenneth. “Bacterial Endotoxin.” Todar’s Online Textbook of Bacteriology: Madison, Wisconsin. 2008. 21 June 2012.   http://textbookofbacteriology.net/endotoxin.html.

 

 

 
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Endotoxin Removal from Biological Solutions

Published on August 16, 2012 by in Home

Endotoxin Removal from Biological Solutions

Summary of Commercial Kits and Services

 

Each removal technique addresses a different problem regarding endotoxins. The most prominent methods that companies have been using are based on separation by size, which uses filtration; separation by charge, which uses ion-exchange resins, separation by phase, which uses a non-ionic detergent Triton X114, and separation by affinity, which uses various ligands.

Commercial Kit Removal Method Recommended Use Company Information
Pierce Detoxi-Gel Polymixin B ligand Proteins Thermo Fisher Scientific
EndoTrapÒ Proprietary non-Polymixin B-based ligand Proteins Hyglos
AltecoÒ LPS Adsorber Tailor-made peptide ligand Proteins Alteco Medical
ToxinEraserTM Modified Polymixin B ligand Proteins Genscript
EtoxiClearTM Proprietary ligand Proteins ProMetic BioSciences Ltd
ProteoSpin Endotoxin Removal Proprietary resin Proteins Norgen Biotek Corp
BcMagTM Quick Endotoxin Removal Beads Polymixin B ligand Proteins BioClone Inc
ProteoSpin Endotoxin Removal Proprietary resin Proteins BioSynthesis
PyroCLEAN Biodegradable detergent Proteins ALerCHEK, Inc
MiroCleanÒ Organic extraction Plasmid DNA Mirus
MoBio UltraCleanÒ Organic extraction Plasmid DNA Mo Bio Laboratories Inc.
Endotoxin Removal Kit Organic extraction Plasmid DNA Lambda Biotek
Endotoxin Removal Kit Detergent partitioning Plasmid DNA BioPioneer
AcrodiscÒ Unit Anion-exchange filters Proteins Pall Corporation
EndotoxinOUT Polymixin B ligand Plasmid DNA G BioSciences
Endotoxin Removal Beads Polycationic ligand Proteins Miltenyi Biotec
Wizard MagneSil TfxTM System Proprietary resin Plasmid DNA Promega
Endotoxin Removal solution Phase partitioning by Triton X114 Plasmid DNA Sigma-Aldrich
Affi-PrepÒ Polymyxin Matrix Polymixin B ligand Plasmid DNA Bio-Rad

Although many researchers rely on various endotoxin removal kits in order to do these processes themselves, there are many factors that can effect endotoxin removal such as ligand density, pH, salt concentration, the isoelectric point, and hydrophobicity of the target molecule.  ARVYS Proteins is a contract research company that offers high-quality endotoxin removal services from protein solutions.  If needed, ARVYS Proteins Inc. will develop customized endotoxin removal protocols when a standard protocol cannot be applied for various reasons.

 

For more information, visit  www.arvysproteins.com

 

 

 

 

 
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Production of Recombinant Proteins in a Soluble Form in E.coli

Published on August 16, 2012 by in Home

Proteins are an essential part of nutrition, medicine, and biochemistry. Modern technology has allowed producing recombinant proteins in large quantities for research and therapeutic use. One of the most common expression systems for protein production is E. coli. However, when heterologous (non-bacterial) proteins are expressed in bacteria, often a produced protein is found in “inclusion bodies,” misfolded or improperly formed protein, as opposed to properly formed, soluble protein. While recovery of a biologically-active protein from inclusion bodies is feasible through refolding, protein expression in a soluble form is still highly desirable.  Thus, efforts for optimization of soluble expression in bacteria are an important part of protein production project.

The following methods had been used to improve recombinant protein solubility in E.coli:

1- Expression of a protein in a truncated form.

Often large heterologous proteins are difficult to express in E.coli, as they are easily denatured or made insoluble. Thus, the expression of a smaller part of the selected protein can improve solubility.

 

2 – Reduction of the protein synthesis rate.

The rate can be slowed by lowering the growth temperature or the inducer concentration, as well as through the use of a weaker promoter, DNA which initiates transcription of a protein or gene.

 

3 – Alteration of the growth environment.

The regulation of pH fluctuations with a buffer and the addition of co-factors, which allow for protein folding/stability, are common methods. Increasing osmotic pressure through the use of polyols and sucrose allows for the accumulation of compatible solutes in the cells, which stabilize protein structures, allows for more soluble protein to be produced as well.

 

4 – Co-expression of a target protein with molecular chaperones.

Chaperones assist with folding and assembly during protein synthesis. They can significantly improve protein folding within the cells. However, the level of chaperone expression needs to be varied for optimal production.

 

5 – Protein expression in the periplasm or “inner wall zone.”

This allows for the formation of disulfide bonds, which are necessary for disulfide bond containing protein. However, such expression can result in lower yields, while some expressed protein can still be present in the cytoplasm and cytoplasmic membrane.

 

6 – Addition of purification tags or fusion proteins to expression

The use of purification tags allow for improved solubility and the promotion of proper protein folding. However, to prevent poor yields or damage to the protein these tags must be removed after expression. “Affinity tags” are the most commonly used, consisting of a small amino acid sequence, but larger “solubility-enhancing” tags are more effective for improving the solubility of proteins that are harder to express. Fusion proteins allow for the combination of peptides, and are often naturally found on the target protein. They can facilitate protein solubility, detection, and purification. Fusion proteins can also be used as a “tag,” to direct protein to a specific part of the cell.

 

Overall, there are many factors that affect protein solubility. The presence of certain amino acids, the type of bacterial host used for expression, and the growth environment in general can all influence protein soluble expression in E.coli. Depending on the nature of a target protein and its final application requirements, the above listed methods can allow for more soluble protein to be expressed.

 

ARVYS Proteins offers high-quality protein services in the area of bacterial protein expression including the generation of expression constructs in a specified vector, expression host selection, expression screening for multiple protein variants, and optimization of expression conditions.

 
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ARVYS Proteins inc.

Published on May 8, 2012 by in Home

ARVYS Proteins Inc. is a contract research organization that specializes in custom protein services. Arvys offers its protein biochemistry expertise to the life science, biotechnology and pharmaceutical communities. We help customers from any research sector at any stage of their protein research and development project, big or small. Our goal is to be your preferred outsourcing choice for protein biochemistry needs and we work hard to earn your business.

Our expertise in protein expression, purification and characterization assures efficient and economical production of active recombinant proteins. We offer a number of expression systems designed to meet specific goals, ranging from expression of tagged proteins for discovery research to production of biopharmaceuticals for preclinical development. Protein production yields are optimized by selection of an appropriate expression system, generation of a high-producing expression construct, optimization of fermentation/cell culture conditions and purification process. Each project is tailored to the customer’s specifications of scale, purity, analytical characterization and activity.

 
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