Secretory expression systems

Evaluation of Secretory Expression Systems for Pharmaceutical Purposes 5... 

In terms of downstream processing efficiency, secretory expression systems indeed offer potential advantages for production of recombinant proteins compared with inclusion body-forming cytosolic systems, but most of the potentially available secretory systems are not yet fully competitive for high-volume therapeutics like insulin and therefore still require intensive improvement efforts. 

Plant-based production systems are now being used commercially for the synthesis of foreign proteins [1-3]. Post-translational modification in plant cells is similar to that carried out by animal cells plant cells are also able to fold multimeric proteins correctly. The sites of glycosylation on plant-produced mammalian proteins are the same as on the native protein however, processing of N-linked glycans in the secretory pathway of plant cells results in a more diverse array of glycoforms than is produced in animal expression systems [4]. Glycoprotein activity is retained in plant-derived mammalian proteins. 

A significant observation from our study of the sTF expression system is that the bacterial secretory machinery continues to function in the presence of Trp analogs. We find similar ratios of total protein produced in the media for Trp, 7-ATrp, and 5-OHTrp (Table I), and spectral evidence for analog incorporation in all media and cell fi-actions that we tested (Fig. 2 c and 2 d)... 

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... 

Expression of recombinant aprotinins has been achieved in E. coli K12 as a fusion with parts of the MS-2 polymerase gene [16]. In this case the fusion protein is deposited as inclusion bodies. Functionally active aprotinin can be obtained after solubilization and purification of the fusion protein, CNBr (cyanobromide) cleavage and renaturation of the aprotinin moiety. Low-level periplasmic expression of native, properly folded aprotinin has been shown in E. coli employing the E. coli alkaline phosphatase signal sequence [17]. With respect to expression level and ease of purification, it transpired that secretory expression in the yeast Saccharomyces cerevisiae is by far the most attractive system for the production of this aprotinin variant [18]. In addition, due to the absence of an N-glycosylation site there are no problems with non-human glycosylation of the protein in yeast. 

In conclusion, the described yeast system is well suited for the secretory expression of small disulfide-bonded, non-glyco-sylated proteins. It should be emphasized that the performance of yeast cells for expression of a particular protein can be further optimized by conventional strain development programs using UV-irradia-tion or treatment with mutagenic compounds. 

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... 

ENHANCEMENT OE PRODUCTIVITY 1.1.3.1 Secretory Recombinant Expression Systems... 

CNTF is expressed in glial cells within the central and peripheral nervous system. CNTF lacks a signal sequence and is not secreted by the classical secretory pathway, but is thought to convey its cytoprotective effects after release from adult glial cells by some mechanism induced by injury [3,5]. 

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