System expression

Several expression systems were first developed in L. lactis in order to express and control heterologous protein production (Bermudez-Humaran 2009). To do so, several constitutive promoters were initially isolated from a genomic library (van der Vossen et al. 1987) with varying protein expression levels (de Vos et al. 1999). However, expression of constitutive protein may lead to a cytoplasmic accumulation. 

Nisin-controlled gene expression (NICE) The NICE system is the most commonly used inducible expression system for heterologous proteins production in LAB. The synthesis. 

Zinc-inducible expression systems (P j ZitR-driven Heterologous Expression and Zirex)  

More recently, a new zinc-inducible expression system, called Zirex, was developed for L. Metis (Mu et al. 2013). When triggered by a non-toxic concentration of zinc, the streptococcal promoter PczcD (regulated by the SezA repressor) is activated and leads to elevated expression levels. Moreover, Mu and colleagues have successfully combined both the NICE system and Zirex to produce different proteins at different times in the same microorganism. 

Self-regulated promoters by environmental conditions A new generation of inducible promoters has been developed to dispense with the use of inducers. Indeed, for in vivo expraiments, LAB harboring expression systems such as NICE, XIES, or SIP need to be induced beforehand 

An interesting and more recent expression system for the development of cell-based assays is based on BacMams. These recombinant baculoviruses containing mammalian cell-active expression cassettes seem to be an efficient strategy to speed up assay development. These viruses are produced in insect cells and transiently express but do not replicate in transduced mammalian cells. The expression level can be well controlled by titrating the amount of virus. Highly reproducible transient expression levels, which are a prerequisite to use transient transfection for HTS, might thus be an attractive alternative for some approaches. 

Comparison between different systems for transmembrane protein expression are always difficult to make, and they obviously reflect individual needs and are strongly influenced by personal experience. It is, however, important to define the usefulness of each system by taking into account different aspects such as ease of handling, expression levels, time and labor requirements, safety, costs, and the quality of the produced recombinant protein (Tab. 1.3). 

Obviously, prokaryotic systems are easy to use, the costs for their large-scale applications are low, and no safety risks are involved. The drawbacks are their limited capacity for post-translational modifications and generally low yields of complex mammalian 

Vector Handling Expr. /Scale-up Authenticity Time/Labour Safety Costs 

Halobacterium salinarum Easy Low/easy Low Short/easy High Low 

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Stability and Purification of the Recombinant Protein. There are no hard and fast rules specifying, eg, whether a recombinant protein is available in a soluble state in the cell. In some cases, the expression system must be engineered by in vitro mutagenesis to optimize overall yield of the protein. 

Gene Expression Systems. One of the potentials of genetic engineering of microbes is production of large amounts of recombinant proteias (12,13). This is not a trivial task. Each proteia is unique and the stabiUty of the proteia varies depending on the host. Thus it is not feasible to have a single omnipotent microbial host for the production of all recombinant proteias. Rather, several microbial hosts have to be studied. Expression vectors have to be tailored to the microbe of choice. 

NMD A receptors are selectively activated by A/-methyl-D-aspartate (NMD A) (182). NMD A receptor activation also requires glycine or other co-agonist occupation of an allosteric site. NMDAR-1, -2A, -2B, -2C, and -2D are the five NMD A receptor subunits known. Two forms of NMDAR-1 are generated by alternative splicing. NMDAR-1 proteins form homomeric ionotropic receptors in expression systems and may do so m situ in the CNS. Functional responses, however, are markedly augmented by co-expression of a NMDAR-2 and NMDAR-1 subunits. The kinetic and pharmacological properties of the NMD A receptor are influenced by the particular subunit composition. 

With few exceptions, information on the anticonvulsant pharmacology of specific ion channel subunits analyzed in expression systems is scarce. Hitherto, a first understanding of the mechanism of action of most antiepileptic dtugs has evolved from analyses of somatic ion channel pharmacology either in isolated neurons from human or rodent neurons, or cell culture models. 

Heterologous expression systems comprise prokaryotic organisms (e.g., E. coli) and eukaryotic cells (e.g., yeast, HEK293, Xenopus oocytes), which are used to functionally express foreign genes or cDNAs. 

Receptor subtype Expression system Transduction pathway Agonist Antagonist Inverse agonist... 

Heparin Sulfate Proteoglycans Hepatic Lipase Hepatitis Hepatitis C Heptahelical Domain Heptahelical Receptors HERG-channels Heterologous Desensitization Heterologous Expression System Heterotrimeric G-Proteins Hidden Markov Model High-density Lipoprotein (HDL)... 

This suite of BVMOs is available via whole-cell expression systems and represents a complementary platform of biocatalysts for diverse applications in chiral synthesis. Representatives of this collection were utilized in the enantiodivergent synthesis of the indole alkaloids alloyohimbane and antirhine from a fused bicyclic precursor (Scheme 9.19) [151]. 

In particular, the availability of such bacterial biocatalysts in the form of recombinant expression systems [136] in combination with simplified purification protocols opened up this methodology for large-scale applications [204]. 

The ELP expression system was compared to the conventional oligohistidme fusion, which is traditionally applied for purification by immobilized metal affinity chromatography (IMAC). Both techniques were shown to have a similar yield of the recombinant protein. The temperature-triggered approach offers a fast and inexpensive nonchromatographic separation with the possibility for larger scale purification. Although the ELP expression system may not be applicable to all types of recombinant proteins, numerous examples have already been shown [40]. 

Actinomycetes Large surface area to volume ratio should favour protein export Widely used in industrial microbiology Good expression systems being developed Promoters/gene regulation still poorly understood Rheology of fermentations important... 

Mammalian cells Get export of proteins Get desired post-translational modifications and products not likely to be immunogenic to humans Good expression systems available Large-scale growth of animal cells costly Great care needed to avoid contamination of cultures... 

Protein Size/struoture Expression system Clinical indications Comments... 

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