This situation may be regarded as the worst case scenario. When the protein of interest cannot be detected through a sensitive technique(e.g.,Western blot) or it is detected but at very low levels(less than micrograms per liter of culture), the problem often lies in a harmful effect that the heterologous protein exerts on the cell.
The problem of protein toxicity may arise when the bacterial protein performs an unnecessary and detrimental function in the host cell. This function interferes with the normal proliferation and homeostasis of the micro organism and the visible result is slower growth rate, low final cell density, and death.
Protein toxicity adversely affects the cloning and expression. For example, the genes or cDNAs of the most toxic proteins are difficult to be cloned. Protein toxicity is the most important reason for DNA cloning or subcloning problems. Most expression problems are also the result of protein toxicity. With optimization of expression vectors and host cells, we estimate that 80% protein yield problems are the caused by protein toxicity. Only 20% yield problems are the results of other reasons such as codon usage. This is why many codon optimized genes still have expression problems.
Codon bias: Although several codons code for a single amino acid, an organism may have a preferred codon for each amino acid. This is called codon bias. Codon bias index is another measure of directional codon bias, it measures the extent to which a gene uses a subset of optimal codons. CBI is similar to Fop, with expected usage used as a scaling factor. In a gene with extreme codon bias, CBI will equal 1.
The decline in codon bias is especially significant for several reasons. First, since the frequency of codon usage is positively correlated with tRNA availability in the cell, the increased use of rare codons is expected to negatively affect protein translation rates.
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
Slower rates of protein production give newly transcribed recombinant proteins time to fold properly.This was previously addressed when we discussed the role of translational pauses at rare codons and the irimpact in the production of recombinant proteins.Moreover, the reduction of cellular protein concentration favors proper folding. By far,the most commonly used way to lower protein synthesis is reducing in cubation temperature Low temperatures decrease aggregation, which isfavored at higher temperatures due to the temperature dependence of hydrophobic interactions.
When IB formation is a problem, bacterial protein production should be carried out in the range15–25◦C, though one report described successful expression at 4◦C for 72h. However, when working at the lower end of the temperature range, slower growth and reduced synthesis rates can result in lower protein yields. Also, protein folding may be affected as the chaper one network may not be as efficient. The Artic ExpressTM (Stratagene) strain(Bline) possesses the cold-adapted chaperonin Cpn60 and co-chaperon in Cpn10 from the psychrophilic bacterium Oleispira antarctica. The chaperonins display high refolding activities at temperatures of 4–12◦C and confer an enhanced ability for E. coli to grow at lower temperatures.
Bacterial Endotoxin Definition: 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) andimmunogenicity 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.
Endotoxins are mostly found in the outer membrane of Gram-negativebacteria. They are the integral part of the outer cell membraneand are responsible for the organization and stabilityof the bacteria. The general structure of all endotoxinsis a polar heteropolysaccharide chain, with three distinctdomains: the O-antigen region, a core oligosaccharide partand a Lipid A part.
Lipid A is the most conserved part which is responsiblefor the toxicity of endotoxins, while, the effect of polys accharides is negligible. The Lipid A structures werefirst studied based on Enterobacteria. The common architecture of Lipid A is a disaccharide, with glucosamine being the monomer. The two glucosamine monomers arelinked between position 1 and 6, and both of themare phosphorylated to produce bisphosphorylated β-(1-6)-linked glucosamine disaccharide. Furthermore, there arefatty acids ester-linked at positions 3 and 3 and amide linkedat positions 2 and 2. The position 6 is attached to theoligos accharide region.
The oligos accharide moiety is the core unit of LPS. Enteric bacterial LPS cores typically consist of 8–12 sugarunits. Alternative structures are reported for the innercore where the heptose may be substituted by a phosphate,pyrophosphate, or phosphory lethanolamine group. The phosphate groups and charged sugar residues in the innercore and Lipid A are responsible for the stability of LPS byinteractions with cations. Moreover, a diversity of negative lycharged components is also reported, such as one to threeunits of α-3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and hexuronic acid.
The O-specific chain is composed of repetitive subunitsand only exists in smooth-type Gram-negative bacteria.There may be up to 50 identical subunits in an O-chain unit,and each subunit consists of up to eight sugar units. Unlikethe inner core region, the frequent components in O-chainstructures are deoxysugars. There are various O-chainstructures, including linear or branched backbones which aresubstituted by many kinds of aglycones. The O- and Nacetylphosphate and phosphorylethanolamine are commonsubstitutes found. Some non-stoichiometric substitutes mayalso exhibit, such as amino acids, acetamidino groups as wellas formyl groups.
From: Chromatographic Removal of Endotoxins: A Bioprocess Engineer’s Perspective
The physiological activities of LPS are mediated mainly by the Lipid A component of LPS. Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system. During infectious disease caused by Gram-negative bacteria, endotoxins released from, or part of, multiplying cells have similar effects on animals and significantly contribute to the symptoms and pathology of the disease encountered.
Since Lipid A is embedded in the outer membrane of bacterial cells, it probably only exerts its toxic effects when released from multiplying cells in a soluble form, or when the bacteria are lysed as a result of autolysis, complement and the membrane attack complex (MAC), ingestion and killing by phagocytes, or killing with certain types of antibiotics.The injection of living or killed Gram-negative cells or purified LPS into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions, such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shockand death. Injection of fairly small doses of endotoxin results in death in most mammals.
Endotoxin Removal service
During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial expression systems remain most attractive due to low cost, high productivity, and rapid use. Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins.
– bacterial expression system introduction from:
E.coli is one of the most widely used bacterial expression hosts, and DNA is normally introduced in a plasmid expression vector. The techniques for overexpression in E.coli are well developed and work by increasing the number of copies of the gene or increasing the binding strength of the promoter region so assisting transcription.
Non-pathogenic species of the gram-positive Corynebacterium are used for the commercial production of various amino acids. The C. glutamicum species is widely used for producing glutamate and lysine, components of human food, animal feed, and pharmaceutical products.
Unlike gram-negative bacteria expression systems, the gram-positive Corynebacterium lack lipopolysaccharides that function as antigenic endotoxins in humans.
The non-pathogenic and gram-negative bacteria expression systems, Pseudomonas fluorescens, is used for high level production of recombinant proteins; commonly for the development bio-therapeutics and vaccines. P. fluorescens is a metabolically versatile organism, allowing for high throughput screening and rapid development of complex proteins. P. fluorescens is most well known for its ability to rapid and successfully produce high titers of active, soluble protein.
– bacterial expression system category from: