Endotocxin Definition And Removal

Endotoxin Introduction

Lipopolysaccharides (LPS), also known as lipoglycans and endotoxins, Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. Although the term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens such as Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis and Vibrio cholerae.

The relationship of endotoxin (lipopolysaccharide) to the bacterial cell surface

The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory responses in an animal and it activates complement by the alternative (properdin) pathway, so it may be a part of the pathology of Gram-negative bacterial infections.

From: http://textbookofbacteriology.net/endotoxin.html

Structure of Endotoxins

Endotoxins are mostly found in the outer membrane of Gram-negative bacteria. They are the integral part of the outer cell membrane and are responsible for the organization and stability of the bacteria. The general structure of all endotoxins is a polar heteropolysaccharide chain, with three distinct domains: the O-antigen region, a core oligosaccharide part and a Lipid A part.

Lipid A is the most conserved part which is responsible for the toxicity of endotoxins, while, the effect of polysaccharides is negligible. The Lipid A structures were first studied based on Enterobacteria. The common architecture of Lipid A is a disaccharide, with glucosamine being the monomer. The two glucosamine monomers are linked between position 1 and 6, and both of them are phosphorylated to produce bisphosphorylated β-(1-6)- linked glucosamine disaccharide. Furthermore, there are fatty acids ester-linked at positions 3 and 3 and amide linked at positions 2 and 2. The position 6 is attached to the oligosaccharide region.

From: Chromatographic Removal of Endotoxins: A Bioprocess Engineer’s Perspective

Endotoxin Removal

BIC offers one-stop endotoxin removal service including gene synthesis, protein expression and purification, and endotoxin detection and removal. More details from: http://www.biologicscorp.com/blog/bacterial-endotoxin-definition/

High Cell Density Fermentation Definite

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 cell density fermentation VS Large scale fermentation（Picture 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.

Notes:

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.

TROUBLESHOOTING BACTERIAL PROTEIN EXPRESSION

Problem	Possible explanation	Solutions
No or low expression	Protein may be toxic before induction	Control basal induction l Add glucose when using expression vectors containing lac-based promoters l Use defined media with glucose as source of carbon l Use playsS/playsE beaning strains in T7-based systems l Use promoters with tighter regulation
	Protein may be toxic after induction	Lower plasmid copy number Control level of induction l Tuneable promoters l Use strains that allow control of induction [Lemo21(DE3)strain] or lacY − strains (Tuner™)
	Codon bias	Lower plasmid copy number Use strains that are better for the expression of toxic proteins(C41 or C43) Direct protein to the periplasm Optimize codon frequency in cDNA to better reflect the codon usage of the host Use codon bias-adjusted strains Increase biomass: l Try new media formulations l Provide good aeration and avoid foaming
Inclusion body formation	Incorrect disulfide bond formation	Direct protein to the periplasm Use E. coli strains with oxidative cytoplasmic environment
	Incorrect folding	Co-express molecular chaperones Supplement media with chemical chaperones and cofactors Remove inducer and add fresh media Lower production rate: l Lower temperature. If possible, use strains with cold-adapted chaperones l Tune inducer concentration

Information from: Recombinant protein expression in Escherichia coli

Inclusion Bodies Formation

The formation of inclusion bodies may proceed through nonpermissive pathways from folding intermediates during the folding process as suggested by Mitraki and King, and temperature is one parameter that affects the conformation and stability of proteins. Increased temperature has been found to stimulate aggregation in several cases. The effect of the induction temperature on the formation of inclusion bodies of SpA-Pgal also indicates that the formation might be caused by hydrophobic interactions between protein chains. A higher temperature increases hydrophobic interaction and might also expose hydrophobic stretches of amino acids that are normally not exposed. The lack of inclusion body formation after changes in the amino acid sequence in the linker region between protein A and 3-galactosidase also indicates that the folding of SpA-Pgal may be important for the formation of a soluble protein. An alternative explanation would be specific intermolecular

interactions of the linker region of SpA-Pgal from pRITL. Thirty-eight of the amino acids in the linker region originate from the C terminal of the lac repressor protein, Lacd. The C terminal of Lacd is not involved in DNA binding but might be involved in binding of inducer or in the formation of its tetrameric structure. The linker region of pRITl-encoded SpA-Pgal is exposed since it is subjected to proteolytic cleavage in the linker by the outer membrane-bound protease OmpT during purification. The linker also contains regions rich in hydrophobic amino acids. Lee and coworkers have shown that when a hydrophobic sequence is introduced between P-galactosidase and a region of the hepatitis B virus surface antigen, inclusion body formation increases with the incubation temperature. The fusion protein without the hydrophobic sequence remained soluble independent of the incubation temperature.

From: Factors Influencing Inclusion Body Formation in the Production of a Fused Protein in Escherichia coli

Bacterial expression system technology platform guarantee protein expression yield and success rate, tech support include:

1. Codon and mRNA structure optimization
2. Vector construction
3. Multiple purification methods
4. Multi-channel protein refolding
5. MALDI-TOF
6. Protein N-terminal sequencing
7. Aliquot and lyophilisation

More information from: http://www.biologicscorp.com/bacterial-system

Protocol For Bacterial Protein Expression And Purification

Bacterial protein expression and purification

Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (protocol for how to make competent cells.)

1.Take competent cell stock aliquot (about 100 mL) out of -80℃ freezer. Place on ice to thaw about 5 minutes.

2.Take one mL of plasmid DNA (usually a pRSET B vector with your protein cloned inside DNA seq of pRSET B) and add to the thawed competent cells. Vortex briefly to mix, and let incubate on ice for at least 20 minutes. Longer incubation will not hurt.

3.Heat shock the competent cells at exactly 42℃ for 1 minute. Place on ice.

4.Add heat shocked cells to 1 mL of LB broth in a sterile culture tube, but DO NOT ADD antibiotic. Shake at 37℃ for at least 1 hour. Shaking for 3 hours doesn't seem to hurt.

5.Plate the cell culture onto LB agar plates (usually 100 mL of culture will do although it depends on the competency of the cells and the initial concentration of plasmid). Make sure the plates have the appropriate antibiotic (usually ampicillin at 100 mg/ml concentration). (protocol for how to make LB Ampicillin Agar Plates) Incubate the plate at 37℃ for 14 hours (overnight generally). Longer times of incubations will allow for satellite colonies to appear.

Step 2: Make a starter culture for protein expression. Usually to about 250 to 500 mL of LB broth the antibiotic is added.

1. Generally, the antibiotic is ampicillin, which is added to the LB broth at 100 mg/ml final concentration. (Have a stock of ampicillin in the 4℃ which is 1000x concentrated or 100 mg/ml in sterile distilled water to make things easier).

2. Pick a colony or two from your plate using a sterile innoculation loop and add to the LB broth/ampicillin flask. Incubate in a shaker (250 RPM) at 37℃ until the OD @ 600 nm is 0.5 to 1.5 OD. (Anywhere in this range is fine, however, extremely dense starter cultures>1.5 OD can cause problems in protein expression. Under these dense growth conditions the plasmid seems to be rejected or turned-off by BL21(DE3)s.)

3. Save your starter culture flask by placing in the 4℃ overnight. This will slow down the bacterial growth enough, but prevent the problems dense cultures cause as listed above.

Step 3: Make the big batch of bacteria culture for protein expression. (How many 1.5 L volumes should you grow up? Expect about 100 mgs of protein per liter, but that estimate is very dependent on the protein!)

1. Equally divide your starter culture among several 1.5 L volumes of sterile LB broth in 2.8 L Fernbach culture flasks. (Good precision when equally dividing the starter will better control the timing when induction can occur.)

2. Add antibiotic to 100 mg/ml final concentration. Grow them up in a New Brunswick shaker large enough to hold several Fernbach flasks.

3. Grow until an OD 600 nm of 0.8 to 1.2. Then induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. This is expensive stuff so use it carefully. Only measure out exactly the amount of IPTG you need for your flasks. Dissolve that amount in about 10 ml of sterile water and divide it equally among your 1.5 L flasks. This stage is called induction. Keep the culture shaking at 37℃.

4. Induction of protein generally takes 3 to 4 hours (but this too depends on your protein). After induction, centrifuge your bacteria in 500 ml bottles in the big Sorvall rotor at 5,000 RPM (bigger than the GSA found on the 2nd floor with Rice, Correl, and Moffat Labs). Do this in batches until all your culture is spun down saving the cell pastes each time. Freeze the cell paste at -80℃.

Step 4: Lysis and sonication of the bacteria. There are many ways to lyse bacteria, skin chickens etc., but this method is tried and true.

1. Make Lysis Buffer: 25 mM TRIS-Cl, 2 mM EDTA, pH 7.6. (You may add protease inhibitors like benzamidine or PMSF if you like but add these right before you start to lyse. Make concentrated PMSF solutions in pure ethanol since it is hydrophobic.) Then add lysozyme at 100 mg/ml concentration (or just a tipful from a spatula). Generally the volume lysis buffer is 1/20 to 1/50 the volume of the bacterial culture.

2. Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 ml pipet or whatever means necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.

3. In order to eliminate the extreme turbidity of the suspension, sonicate the suspension to shear the DNA until the turbidity is similar to that of a normal protein solution. Then centrifuge at 18,000 RPM in a Sorvall SS-34 rotor (or ~40,000 x g) for 20 minutes at 4℃.

4. Save the pellet and the supernatent. (If the solution is slighly turbid due to residual DNA, a quick way to shear the DNA is to pass through a syringe with a needle.) Generally your protein is in the supernatent so simply freeze your pellets until you know where your protein is. Protein purification from this supernatent, however, will depend on the properties of the protein: its isoelectric point (pI), size, hydrophobicity, etc.

BiologicsCorp offers high-level E. coli protein expression services and technology platforms to provide products for your concerns.

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More details: bacterial expression system