Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.

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Secretory and extracellular production of recombinant proteins using Escherichia coli

http://wolfson.huji.ac.il/expression/local/bacteria/Recombinant%20protein%20secretion%20in%20Escherichia%20coli-2005.pdf

Recombinant protein secretion in Escherichia coli.

Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein's over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.

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Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies.

Numerous studies have been published that attempted to correlate fructan concentrations with freezing and drought tolerance. Studies investigating the effect of fructan on liposomes indicated that a direct interaction between membranes and fructan was possible. This new area of research began to move fructan and its association with stress beyond mere correlation by confirming that fructan has the capacity to stabilize membranes during drying by inserting at least part of the polysaccharide into the lipid headgroup region of the membrane. This helps prevent leakage when water is removed from the system either during freezing or drought. When plants were transformed with the ability to synthesize fructan, a concomitant increase in drought and/or freezing tolerance was confirmed. These experiments indicate that besides an indirect effect of supplying tissues with hexose sugars, fructan has a direct protective effect that can be demonstrated by both model systems and genetic transformation.

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Fructan and its relationship to abiotic stress tolerance in plants

Plants are sessile and sensitive organisms known to possess various regulatory mechanisms for defending themselves under stress environments. Fructans are fructose-based polymers synthesized from sucrose by fructosyltransferases (FTs). They have been increasingly recognized as protective agents against abiotic stresses. Using model membranes, numerous in vitro studies have demonstrated that fructans can stabilize membranes by direct H-bonding to the phosphate and choline groups of membrane lipids, resulting in a reduced water outflow from the dry membranes. Inulin-type fructans are flexible random-coiled structures that can adopt many conformations, allowing them to insert deeply within the membranes. The devitrification temperature (Tg) can be adjusted by their varying molecular weights. In addition, above Tg their low crystallization rates ensure prolonged membrane protection. Supporting, in vivo studies with transgenic plants expressing FTs showed fructan accumulation and an associated improvement in freezing and/or chilling tolerance. The water-soluble nature of fructans may allow their rapid adaptation as cryoprotectants in order to give optimal membrane protection. One of the emerging concepts for delivering vacuolar fructans to the extracellular space for protecting the plasma membrane is vesicle-mediated, tonoplast-derived exocytosis. It should, however, be noted that natural stress tolerance is a very complex process that cannot be explained by the action of a single molecule or mechanism.

/pdf/plant-fructans-in-stress-environments-emerging-concepts-and-276sws7vuv.pdf

Plant fructans in stress environments: emerging concepts and future prospects

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l−1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 106), as determined by HPLC while high molecular weight levan (>6 × 106) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

Chitinases help degradation of cell walls containing chitin and thus accelerates protoplast formation. It indirectly helps strain improvement and development of new strains which are economically viable for industrial use. Chitinases consist of endochitinase, exochitinase, β-N-acetylglucosaminidases and chitobiases. The endochitinases can be further characterized for better understanding of mechanism of the enzyme reaction. This review covers the recent advances in isolation and characterization of endochitinases and its components, gene encoding sequences and cloning.

Ki Bang Song

Papers

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

Characterization and applications of chitinases from Trichoderma harzianum – A review

Characteristics of levan fructotransferase from Arthrobacter ureafaciens K2032 and difructose anhydride IV formation from levan.

Levansucrase of Rahnella aquatilis ATCC33071: Gene Cloning, Expression, and Levan Formation

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli.