Host-vector system

The cell or microorganism before harbouring the vector is referred to as the host cell, and the stable association of the two used in the manufacturing process is referred to as the host-vector system. 

The suitability of the host-vector system, particularly as regards microbiological purity, is demonstrated by ... 

DNA probes. Purified DNA is obtained from the host-vector system grown under the same conditions as those used in the manufacturing process. Host chromosomal DNA and vector DNA may be separately prepared and used as probes. 

Both H- and L-NHase genes are expressed under the control of a lac promoter in E. coli only when the transformants are cultured in the presence of CoCl2 [63], However, the level of NHase activity in their cell-free extracts is much lower than those of H- and L-NHases in R. rhodochrous Jl. Most of H-NHase is produced as an insoluble form in the E. coli transformant, and there is only a little L-NHase in either the supernatants or precipitates of the extracts. Establishment of an effective host-vector system in Rhodococcus [71] facilitates the development of strains with improved NHase activities. In this connection, the transformation system in Rhodococcus has been investigated. Enzyme assays of recombinant Rhodococcus cells showed that a downstream region of the Rhodococcus sp. N-774 NHase gene is indispensable for the production of active... 

A. Plasmid Construction and Establishment of Host-Vector System in S. fradiae... 

The selected production procedure affects the nature and level of potential contaminants. The possible variability of the system during production may result in modifications that favor the expression of alternative genes in the host-vector system or produce lower product efficiency, or quantitative and qualitative differences in the present impurities. An example of such variability is a change in the profile of proteases... 

The maximum length of a continuous culture should be based on information about the system and product uniformity and stability. In long continuous cultures, the cell line and product will be repeatedly evaluated at intervals determined by the information on the stability of the host-vector system and the product characteristics. 

Protein microsequencing, peptide synthesis Site-directed mutagenesis Creation of specific mutant proteins Protein overproduction Appropriate host/vector systems... 

Two factors which dictate the choice of a host/vector system are the ability to produce the protein in a form acceptable for the particular application, and the economics of production. While biological activity of the protein is the criterion for acceptability in most cases, in some applications such as vaccines, the protein is useful irrespective of its biological activity. Evaluation of the economics should include not only the cost of growing the engineered cell, but also the downstream purification techniques necessitated by the characteristics of the production system. Table 2.9 is a list of the expression systems currently in use, and their advantages and disadvantages. 

CELL TYPES—CHOOSING A HOST/VECTOR SYSTEM... 

Table 1 Comparison of cell host-vector systems for product synthesis...

Because CCT is assembled from eight different polypeptides, the prospect of engineering a host/vector system for the expression of recombinant chaperonin is a daunting one hence, all studies of CCT have thus far depended on material purified from a eukaryotic source such as mouse or bovine testis or rabbit reticulocyte lysate. Even if sufficient material could be purified from these tissues, the heterooligomeric nature of the particle might make crystallization extremely challenging. Structural analyses of CCT have therefore been confined to studies by cryo-electron microscopy. Nonetheless, such analyses have proved very useful in identifying some of the unique characteristics of this chaperonin. 

The host-vector system of B. brevis has advantages of high secretion yields of the heterologous proteins as well as very low proteolytic activity of the host [27]. These properties are intrinsic in the bacterium, which was isolated as the strains for protein fermentation by Udaka [71], actually as an extension of Udaka s famous invention of the amino acid bioprocess. One of the selected strains,... 

Sano K (1994) Host-vector systems for amino acid-producing coryneform bacteria. In Recombinant microbes for industrial and agricultural applications. Marcel Dekker, p 485... 

Today, recombinant protein production involves many options. In addition to E. coli, several yeast systems (see Part IV, Chapter 13), insect cells (see Part IV, Chapter 14), different mammalian expression systems (CHO, BHK, NSO, HKBll, PER.C6) (see Part II, Chapter 3 and Part IV, Chapters 1 and 3) other alternative expression systems are currently under development for the production of biopharmaceuticals. These include transgenic animals or plants, and will be discussed in Part IV, Sub-Part 2 of this book. This chapter will focus on E. coli, a still-modern secretory Saccharomyces ccrevisiac system, and the recently developed mammalian HKBll expression system. An E. coli host/vector system is described that was originally developed for the efficient production of an interleukin-4 variant Later, it transpired that this system is ideally suited to the expression of other proteins and Fab fragments. The secretory... 

In conclusion, the described HKBll host/vector system supports the rapid production of milligram and gram quantities of proteins and recombinant antibodies in a large-scale transient transfection format to generate material for proof of concept... 

In the next contribution we learn about hands-on experience and recent improvements with different production systems for biopharmaceuticals at Bayer Health-Care. As previously also published in Nature by Heiner Apeler, Head of Expression, an E. coli host/vector system was originally developed for the efficient production of an interleukin-4 variant, but afterwards it was optimized for the expression of other proteins and even Fab fragments. Process development and optimization of the yeast secretory Saccharomyces cerevisiae for expression of a protease inhibitor will also be presented. The focus, however, is on the use of a recently developed mammalian HKBll (hybrid clone of human kidney and B cells) expression system for recombinant human glycoprotein biopharmaceuticals. HKBll is a favorable cell host for the production of human proteins, because it dehvers biopharmaceuticals that are structurally identical to the natural product. The host/vector system supports the production of gram quantities of proteins in a large-scale transient transfection format as well as the development of stable cell fines. These systems together... 

In order to efficiently and systematically exchange and recombine biosynthetic genes, the biosynthetic genes responsible for various polyketide natural products must be sequenced, and a host-vector system must be available. Several complete biosynthetic gene clusters have now succumbed to cloning and sequencing (7 -84), and a host-vector system has been developed in Strepto-myces (8S). In initial recombination and truncation efforts, compounds exhibiting dramatically different carbon skeletons have been produced. A sample of some of the hybrid structures produced with enzymes taken from the biosynthetic pathways that normally lead to actinorhodin and tetracenomycin (Fig. 15) are shown in Fig. 16. 

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