Current Pharmaceutical Biotechnology - Volume 11, Issue 3, 2010

Biotechnology industries commenced with the production of recombinant proteins for developing biopharmaceuticals. However, scientific development of recombinant technologies has contributed not only to protein pharmaceuticals but also to industrial enzymes, drug development and material sciences. Since the beginning of recombinant technology, numerous pharmaceutical proteins have been developed, covering tissue-type plasminogen activator, insulin, growth hormone, cytokines and various enzymes. Now therapeutic antibodies of both antagonistic and agonistic activities, receptors and various forms of biological modifiers occupy a large part of biopharmaceutical pipelines. A large fraction, e.g., 60-70 %, of these pharmaceutical proteins is produced in mammalian cells, mostly using Chinese hamster ovary (CHO) cells. So far all mammalian recombinant therapeutic proteins are secreted into the media and captured from the media by various separation technologies. The remaining portion of pharmaceutical proteins is presumably made using microbial cells, in particular Escherichia coli and yeast cells. Expression of pharmaceutical proteins in microbial cells can be directed into either cytoplasmic inclusion bodies or the secretory pathway for soluble, folded proteins. Cytoplasmic soluble expression has seldom been used for pharmaceutical proteins, although it is often used to produce reagent proteins. Cytoplasmic soluble expression has some disadvantages for pharmaceutical proteins, e.g., insufficient expression level, heterogeneous N-terminal processing and proteolytic cleavage. Considering the relative ease of cytoplasmic expression as inclusion bodies, there will be an opportunity for technology improvements in the refolding of insoluble proteins. Technological improvement of mammalian expression, including gene expression, host cell engineering, culture medium development and cell culture engineering, now made possible the expression of 10 g/L or higher in cell culture medium, as summarized in Chapter-1. It appears that more improvements will be made to increase the quality of products, which should make downstream capture, purification and separation process more efficient. Conversely, cytokines, intracellular signaling proteins, viral proteins and fragments or domains of such more complex proteins as receptors, antibodies and cell membrane-associated proteins may benefit from the development of novel nonmammalian expression system, which is of low cost and high efficiency. These proteins can be the target of drug candidates, vaccines, pharmaceutical proteins, antigens for pharmaceutical and diagnostic antibodies and reagents for cell culture. For example, recently recombinant proteins, upon delivery into cells, have been shown to reprogram the cells into pluripotent stem cells [1, 2]. Chapter-2 describes the transient mammalian expression of interleukins using fusion partners that help to enhance soluble protein expression. Non-mammalian expression systems include bacteria, yeast, other microbes and insect cells. Expression in these systems leads to either soluble protein or insoluble inclusion bodies (IBs). In both cases, numerous problems are often encountered, e.g., low expression, expression of soluble, but inactive proteins, or low yield following refolding. In these areas, there is always need for technical improvement. In fact, a topic covering “proteins difficult to express” or “proteins difficult to make” frequently appears in the scientific and commercial conferences. We have assembled various novel technologies that cover production of recombinant proteins that are “difficult to express” or “difficult to make” as introduced above. Recombinant expression may be formally divided into soluble and insoluble expression. Insoluble expression primarily occurs in cytoplasmic expression of Escherichai coli (E. coli), yeast and insect cells. When expressed proteins are directed to periplasmic space using a signal sequence, the transported proteins may have correct disulfide bonds, but may still form IBs. In this case, oxidative refolding is not required. Only solubilization is necessary. Since the cell wall of E. coli is permeable to low molecular weight compounds, folding or high solubility may be assisted by adding folding or solubility-enhancing additives into the culture media [3-5]. Soluble expression can occur four different ways. The most familiar one is extracellular expression using mammalian, yeast, insect or bacterial system. Periplasmic expression can also lead to soluble expression. Intracellular proteins can be expressed as soluble in cytoplasm of various host cells, or using cell-free system. When soluble expression of target proteins is difficult, one of the conventional approaches has been to fuse them with a soluble and stable fusion partners [6, 7]. Chapter-2 describes a transient mammalian expression of interleukins using two fusion partners. An unusual example of the fusion technology is with viral polyhedrin protein, which leads to formation of viral capsids containing a fusion of polyhedrin and target protein in the cytoplasm of host cells [8]. The expressed target proteins may be used as attached to viral capsids or cleaved from the polyhedrin proteins. Insect cells are also commonly used as a host cell for extracellular expression. A new host-vector system was developed for protein expression in Silkworm (Chapter-3). An efficient secretory expression system was developed with Brvebaccilus bacterium as described in Chapter-4. Cytoplasmic soluble expression of foreign proteins is often difficult, leading to formation of inclusion bodies (IBs). An approach often taken is to enhance in vivo folding....

Mammalian cell lines are important host cells for the industrial production of pharmaceutical proteins owing to their capacity for correct folding, assembly and post-translational modification. In particular, Chinese hamster ovary (CHO) cells are the most dependable host cells for the industrial production of therapeutic proteins. Growing demand for therapeutic proteins promotes the development of technologies for high quality and productivity in CHO expression systems. The following are fundamentally important for effective production. 1) Construction of cultivation process. The CHO-based cultivation process is well established and is a general platform of therapeutic antibody production. The cost of therapeutic protein production using CHO cells is equivalent to that using microbial culture. 2) Cell line development. Recent developments in omics technologies have been essential for the development of rational methods of constructing a cell line. 3) Cell engineering for post-translational steps. Improvement of secretion, folding and glycosylaiton is an important key issue for mammalian cell production systems. This review provides an overview of the industrial production of therapeutic proteins using a CHO cell expression system.

The expression of proteins which do not express well on their own can be enhanced by linking them to human serum albumin (HSA) or antibody crystallizable fragment (Fc). The constructs shown here are designed to secrete the proteins after transient transfection of mammalian cell lines. The fusion partners are appended to the N-terminus of the proteins and contain a linker designed to be proteolytically cleaved. Transient transfection and purification protocols are provided as well as experimental results with five interleukins.

While the Baculovirus Expression Vector System (BEVS) mainly uses insect cell lines, such as Sf9 cells, the robust high expression system using silkworm has also been developed. We have further improved technologies for enhancement of virus recombination, reduction of proteolytic degradation and aggregation, and more reliable promoters. These developments made it possible to achieve high and soluble expression of recombinant proteins. We review here such technology developments, advantage of using silkworm and some example applications. There are areas where this technology can be further improved as implicated in the end.

Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (∼30g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants.

A novel expression of recombinant proteins was developed using moderate halophiles that accumulate osmolytes and hence provide cytoplasmic environments where osmolyte-driven folding can take place. Promoters and selection marker were developed for high expression of foreign proteins. Examples are given for expression of bacterial nucleoside diphosphate kinase and human serine racemase.

The PURE system is a highly controllable cell-free protein synthesis system composed of individually prepared components that are required for protein synthesis in Escherichia coli. The PURE system contains neither nucleases nor proteases, both of which degrade DNA or mRNA templates and proteins. The protein products are easily purified using affinity chromatography to remove the tagged protein factors. The PURE system should help to create new fields in protein research.

We have made a dramatic improvement of the wheat cell-free protein synthesis system. The first key improvement is the method for preparation of the cell-free extract that is free of inhibitory factors of translation reaction. Additional improvements include a method for preparation of transcription-ready templates by PCR, an expression vector for the cell-free system, and the “bilayer” mode reaction method that is much more efficient than the batch mode method and at the same time easy to be performed by human hands and by liquid handling machines. We review here the history of the development and describe the protocols for the most handy “bilayer” method and a more efficient but complicated methods. Information on many examples and variations of the wheat cell-free protein synthesis methods already published elsewhere is then provided so that the readers can understand the power and potential applications of the methods.

Cell-free protein synthesis systems offer production of native proteins with high speed, even for the proteins that are toxic to cells. Among cell-free systems, the system derived from insect cells has the potential to carry out posttranslational modifications that are specific to eukaryotic organisms, as occurs in the rabbit reticulocyte system. In this review, we describe development of this insect cell-free system and its applications.

Protein refolding is still on trial-and-error basis. Here we describe step-wise dialysis refolding, in which denaturant concentration is altered in step-wise fashion. This technology controls the folding pathway by adjusting the concentrations of the denaturant and other solvent additives to induce sequential folding or disulfide formation.

Protein refolding using urea gradient size exclusion chromatography (UGSEC) is a new version of conventional SEC refolding, which incorporates urea gradient into the SEC. Operating factors, mainly the urea gradient length and the final urea concentration in the gradient, are discussed in detail. Recent applications of UGSEC, including refolding of recombinant human granulocyte colony stimulating factor by UGSEC, is shown as an example.

We have developed a high-pH, pH-shift refolding “Ph-Fold technology”, both for academic research and industrial protein drug development applications. Using this technology, we were able to refold some “difficult-to-refold” proteins, some of which are proven important drug targets and protein drug candidates. The technology is composed of the initial E. coli production of inclusion bodies, the high pH solubilization/pH shift refolding screening technology, which includes automated pH shift refolding screening, and the methods of testing protein refolding without functional assay. This technology, especially the automated refolding system based on the Ph-Fold technology, may help both academic and industrial research in broadening structural genomic research, functional proteomics, and genomic scale drug development efforts.

Molecular chaperones in living systems inspired us to explore new concepts for assisting protein refolding. The chaperone selectively interacts with a non-native protein by hydrophobic interaction to prevent irreversible aggregation and releases the protein in its refolded form with the aid of ATP and another co-chaperone. Cyclodextrins have been used to simulate the function of the chaperones by controlling the hydrophobic interaction with proteins. In this chapter, we review the cyclodextrin (CD)-related protein refolding systems.

Protein refolding from unfolded state is usually carried out at low temperature to reduce protein aggregation and proteolytic degradation. This review briefly introduces a unique method for the protein refolding via high temperature, typically at above melting temperature of the protein.

It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1α, interferon-γ, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturaing solubilization of some of these proteins we have tested.

Aim: To study the effects of an extract solution from radix curcumae on the expressions of VEGF, COX-2 and PCNA in gastric mucosa of rats during carcinogenesis induced MNNG. Methods: Eighty male Wistar rats were randomly divided into 5 groups: group A, water and normal saline; group B, MNNG and normal saline; group C, MNNG and celecoxib; group D, MNNG and low-dose (1g/ml) radix curcumae steam distillation extract solution; group E, MNNG and high-dose (2g/ml) radix curcumae wet distillation extract solution. In the end of the 40th week, all rats alive were sacrificed and the expressions of VEGF, COX-2 and PCNA in gastric mucosa were determined by immunohistochemistry. Results: The expression levels of VEGF and COX-2 and the optical density levels of PCNA in group C, D and E were remarkably lower than that of group B (P<0.05), while the optical density levels of PCNA in group E were higher than that of group C and D (P<0.05). Conclusions: The distilled extract of curcumae can down-regulate the expressions of VEGF, COX-2 and PCNA in the gastric mucosa of rats during carcinogenesis induced MNNG and can reduce the incidence of gastric caner, suggesting it maybe as a potential chemopreventive agent for gastric cancer.