Expression hosts

Grmpound Native producer Pathway-type (size) Yield References 

Heterologous Expression of the Epothllone Gene Cluster from Sorangium cellulosum in Myxococcus xanthus 

---

Chapters 1-4 serve as an introduction to emerging biocatalysts, modern expression hosts, state of the art of directed evolution, high-throughput screening, and bioprocess engineering for industrial applications. 

Figure 2.1 Decision tree for the choice of a preferable expression host...

E. coli, Bacillus and Pseudomonas present quite a solid base for the production of non-glycosylated proteins. Nevertheless, a number of other prokaryotic expression hosts exist that are not as well established and which show features that are not present in the major expression organisms, and this could be extremely useful for special case proteins. 

This chapter provides an overview of the different expression hosts, systems and strategies available in molecular farming, and discusses their advantages and disadvantages for the production of different types of recombinant proteins. 

Tab. 13.1 Plant expression hosts used for molecular farming - advantages and disadvantages.

In this chapter, we have looked at the properties of different expression hosts and expression systems, and considered some of the available strategies to control transgene expression and protein accumulation. When all these variations are combined, there exists a very diverse range of potential expression platforms that can be used to produce recombinant proteins. The choice depends on many factors, some intrinsic to the plant species or expression system, some dependent on the recombinant pro-... 

Several key issues have to be addressed in the downstream processing of biopharmaceuticals regardless of the expression system. The removal of host cell proteins and nucleic acids, as well as other product- or process-related or adventitious contaminants, is laid down in the regulations and will not differ between the individual expression hosts. The identity, activity and stability of the end product has to be demonstrated regardless of the production system. The need for pharmaceutical quality assurance, validation of processes, analytical methods and cleaning procedures are essentially the same. 

Here, we describe the various alternatives to the use of E. coli expression hosts for heterologous gene expression. Advantages and disadvantages for the different expression systems are discussed, and practical aspects of expression technologies are also described. The feasibility of isotope labeling of recombinantly expressed proteins and their potential use for NMR is also discussed, since the costs and quantities of recombinant proteins produced depend on the system being used. 

If specific amino acid-type labeling is required, the labeled amino acid is added to the fermentation of the expression host (topic 1 above, see Sect. 1.2.3). In this case, a thorough isotope analysis of the expressed protein is advisable prior to NMR spectroscopic investigations. This is preferentially achieved by GC-MS analyses of the hydrolyzed amino acids from the protein product. 

There are two yeast expression hosts that have an established track record for high-level production of heterologous proteins, namely Saccharomyces cerevisiae and Pichia pastoris. HTP expression screening using microplate formats has been reported for both these yeasts by Lang and coworkers (Holz et ah, 2002, 2003 Boettner et ah, 2002). In both cases standard protocols have been miniaturized with cells cultured in either 1.5 ml cultures in 96-deep-well plates for S. cerevisiae or 2 ml cultures in 24-deep-well plates for P. pastoris. Soluble... 

---