Selection of suitable methods for recombinant protein expression

Summary

Recombinant proteins are important tools for studying biological processes. Expression systems are needed for their preparation. The choice of a suitable expression system depends on the properties of the recombinant protein, the intended application of the recombinant protein, and the ability of the system to produce sufficient amounts of protein.

Authors: Burgess et al., Translated by Vivian Chen, this experiment is from "Protein Purification Guide".

Operation method

Selection of suitable methods for recombinant protein expression

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I. Introduction

Selection of the appropriate ugly histone expression method is critical to obtaining the required quantity and quality of rez-histone proteins in a timely manner. Choosing the wrong expression host may result in misfolded or under-expressed proteins. Lack of essential post-translational modifications or inappropriate modifications. Factors to consider when selecting an expression system include the size of the protein, the number of disulfide bonds, the type of post-translational modification required, and the localization of the target protein. The periodic use of purified recombinant protein is also critical in the decision-making process, and there are four main areas of application: structural studies, in vitro activity analysis, use as an antigen for antibody production, and in vivo studies. The purpose of this chapter is to provide guidance to the researcher in selecting an appropriate expression system. However, even with these guidelines, in many cases it is not possible to determine in advance which expression system is the most appropriate, and it is necessary to try several expression systems to find the ideal one.

A large number of expression systems are currently in use in academia and industry. Some of these expression systems are very new and have not been tried enough to evaluate their utility. Moreover, some of the established recombinant protein expression systems (e.g., transgenic animals) are technically challenging, time consuming, and extremely expensive, and thus are not viable options for the average laboratory. For the purpose of this chapter, we will only discuss E. coli, Picrytus, baculovirus/insect cells, and mammalian expression systems (see Chapters 12-15 for a more detailed description of these expression systems). All four types of expression systems have simple and easy-to-understand protocols, are inexpensive to prepare in small quantities, and are readily available from peers or research product companies (e.g., Invitrogen, EMI>Novagen, Stratagene, and P rom ega). In the following, we will briefly describe the characteristics of these types of expression systems and the choices available to them, focusing here on the differences between them. Finally, we will present some strategies to help researchers choose the most appropriate expression system.

E. coli

E. coli was the first host bacterium used for recombinant protein expression, and it is still considered a major tool in the field of protein expression. The short proliferation time of E. coli makes it a simple and fast recombinant protein expression system. Therefore, it takes less than 1 week to evaluate the expression of a recombinant gene in E. coli. Media for cultivating E. coli are inexpensive, and simple and straightforward methods are available for scaling up the production process (see Chapter 12 for details).

In E. coli, recombinant proteins are usually localized in the cytoplasm, but can also be localized in the periplasm, and in a few cases are secreted outside the cell. Proteins localized in the cytoplasm are expressed with the highest efficiency, and their production can typically account for up to 30% of the total biomass (Jana and D e b , 20005). However, overexpression of recombinant proteins may lead to the accumulation of insoluble protein aggregates, resulting in the formation of inclusion bodies Gndusi0nS b0dy). Not only proteins of eukaryotic origin form inclusion bodies, but in a few cases, overexpression of prokaryotic origin proteins, including E. coli, also forms inclusion bodies. In E. coli, the rate of protein translation and folding is almost 10-fold higher than in eukaryotes, which may account for the formation of inclusion bodies in eukaryotic proteins (Andersson et al., 1982; Goustin and W ilt, 1982). In some cases, inclusion bodies greatly impede the acquisition of soluble active proteins.

However, sometimes inclusion bodies can be beneficial in that they are not easily degraded by proteases, are easily concentrated by centrifugation, are seldom contaminated by other proteins, and, with some effort, can be refolded into active soluble proteins (see Chapter 17 for details).

1. Escherichia coli: temperature and molecular chaperones

Several techniques have been used to form as many soluble, correctly folded proteins in the cytoplasm as possible and as few inclusion bodies as possible. The easiest way to do this is to lower the temperature to 15 to 30°C while the protein is being expressed (Sahdevetal., 2008). It is hypothesized that lowering the temperature decreases the rate of protein transcription, translation, and folding, thus allowing the protein to fold correctly (V e r a e ta L , 2006). In addition, studies have shown that lower temperatures also reduce the activity of heatshock protease (Spiess e ta L , 1999). Some researchers have promoted protein solubility by co-expressing molecular chaperones with recombinant proteins in the cytoplasm (Young etal., 2004). The application of this method appears to be protein-specific and therefore requires separate experiments for each target recombinant protein (Baneyx and Mujacic, 2004).

2. e. coli: fusion tags

Fusion of soluble fusion tags (fusion tags) at the N- or C-terminus of recombinant proteins is another way to increase the solubility of many recombinant proteins (Esposito and Chatterjee, 2006). Fusion tags that have been shown to improve the solubility of recombinant proteins include glutathione-S-transferase (GST), thioredoxin, maltose-binding protein (M BP), small ubiquitin-modifying protein (SMP), and the small ubiquitin-modifying protein (SMP). small ubiquitin-modifier (S U M O ) and N us A tag (JV-utilization substrate). The G S T and M B P tags have the added advantage of being used as affinity purification tags. Unfortunately, however, no single tag can be applied to all recombinant proteins, and multiple fusion tags must be evaluated for their ability to promote soluble expression. There are several strategies for removing fusion tags from recombinant proteins, and it is widely practiced to insert a protease cleavage site between the fusion tag and the recombinant protein and then excise it using a specific protease. Sometimes the recombinant protein may become insoluble after removal of the tag, so this approach must be carefully tested.

3. E. coli: Formation of disulfide bonds

For cytoplasmic expression, E. coli is usually unable to promote the correct formation of disulfide bonds for recombinant proteins; the periplasm is usually the only place in E. coli where disulfide bonds can be formed due to the catalytic action of the disulfide oxidoreductase system (Dsb system) (Andersen e t a l., 1997; Bardwell, 1994 ). Therefore, if a recombinant protein is required to form a disulfide bond, it needs to be localized to the periplasm using a cleavable signal peptide (e.g. pelB). However, a major disadvantage of periplasmic expression is that protein expression is greatly reduced. The ability of thioredoxin and glutaredoxin to promote the reduction of cytoplasmic hemicarboxylic acid has been demonstrated by modification of the E. coli genome to disrupt the thioredoxin reductase gene (trrB ) and glutathione reductase gene (glutathione reductase,gene,gene,gene,gene,gene,gene,gene,gene,gene,gene,gene,gene,gene and gene. glutathione reductase gene,g o r), it is possible to create an environment in the cytoplasm that is more conducive to disulfide bond formation (Bessette e ta l., 1999). These genetically engineered modified strains have been commercialized by EMI>Novagen (Origami). If more disulfide bonds need to be formed, recombinant proteins can be fused to thioredoxin and then tabulated in trxB-/gor""E. coli strains (LaVallie et aL , 1993).

4. E. coli: post-translational modifications

Finally, it must be recognized that E. coli has a limited ability to perform posttranslational modification of protein compared to eukaryotes. For example, E. coli does not support enzyme-mediated N-linked glycosylation, N-linked glycosylation, amidation, hydroxylation, myristoylation, palmitoylation, and myristoylation. The following are some of the most common types of sulfation: palmitoylation, palmitoylation, and sulfation.

III. Picrospermum

As another powerful traditional tool for the expression of recombinant proteins, Saccharomyces cerevisiae has been successfully applied to the expression of large amounts of proteins. Enzymes have many of the advantages of E. coli, such as short colonization time and easy manipulation of the genome, as well as the advantages of eukaryotes, including improved protein folding and a high degree of post-translational modification. The first fermenter strain commonly used for recombinant protein expression is SaccAarow^yces ceretw』siae (Strausberg and Strausberg, 2001). However, in the last 15 years, Pz'c/ia and U'folk have become the preferred ferments because of the higher level of expression of recombinant proteins than that of the fermenting yeast (see Chapter 13). Beecham ferments are methyltropic yeasts that utilize methanol as their sole carbon source (Cregg et al., 1985). The growth of Beecham yeast in methanol-containing medium leads to strong induction of transcription of alcohol oxidase (A0X) and dihydroxyacetone synthase genes (Cragg, 2007). These proteins are induced to constitute up to 30% of the total biomass of Beecham fermenters. The utilization of this methanol-dependent gene induction has been achieved by integrating the tightly regulated strong promoter of alcohol oxygenase I (AOXL) into most of the recombinant protein expression plasmids to drive the expression of recombinant proteins (Daly and Haim, 2005). The expression carriers of bicarbonate ferments are integrated into the genome, whereas the expression carriers of fermentation ferments are replicating episomally plasmid-based expression carriers, which are very unstable. It takes 3 to 4 weeks to evaluate the expression of a recombinant gene using bicarbonate ferments. This includes the time required for the conversion of the fermentation, the screening of positively integrated transducers and the expression of the protein. One of the attractive features of Beecham ferments is the extremely high cellular density that can be achieved under suitable culture conditions (Craig, 2007). A cell dry weight density of 120 g/L can be achieved with the use of low-cost cultures of Beecham ferments. It should be emphasized that a low percentage of methanol is required in the induction medium. In large-scale culture, the amount of methanol is of such a magnitude that it poses a fire risk and a higher level of safety measures is required.

Beecham ferments have been used to obtain intracellular and secreted proteins. Like other eukaryotes, it can efficiently form disulfide bonds and has been successfully used to express proteins containing multiple disulfide bonds. In order to facilitate secretion and expression, it is necessary to modify the recombinant proteins to carry signal peptide sequences. The most commonly used signal peptide is the pre-pro sequence of the cr-mating factor of fermentation yeast (Daly and Hearn, 2005). Purification of recombinant proteins from cultured bases is a relatively simple task since Bichromobilin produces only a small amount of endogenous proteins. If the enzymatic degradation of the recombinant protein is severe, it can be expressed by a Pep4 proteinase-deficient bilirubin fermenter strain (Gleeson et al., 1998). This strain has a low activity of vaCU0K peptidase A, which is responsible for activation of carboxypeptidase Y and protease B (proteinase B (BI)).

Yeast has the ability to posttranslationally add glycans (glycans) to specific asparagine residues (N-linkage) and serine/threonine residues (〇■ linkage). The structure of these glycans is very different from that of the glycans added by insect and mammalian cellular modifications. In Baker's yeast, N-linked glycans are of the high-mannose (mannose) type, typically with 8 to 17 mannose, whereas Saccharomyces cerevisiae glycans contain 50 to 150 mannose residues (Celik et al., 2007; Gemmill andTrimble, 1999). Similar to insect and mammalian cells, the consistent sequence of N-linked glycans in ferments is Asn-Xaa-Ser/Thr. Two research groups have fully engineered two strains of Saccharomyces cerevisiae that are capable of producing complex N-linked glycan structures comparable to those found in mammalian cells (Hamilton and Gerngross, 2 07). , 207). However, other researchers can only use strains developed by Roland Contreras' group and must obtain permission from Research Corporation Technologies. The structure of the O■-linked glycans in Bibendum ferments has not been well studied, but it is known that they are formed by the addition of 1 to 4 mannose residues to serine/threonine residues (Goto, 2007; Trimble et al., 20 ○ 4). There have been several reports that certain specific proteins are expressed in Picot yeast with the addition of O-linked glycans, whereas endogenous expression in mammalian cells is not (Daly and Hearn, 2005).

iv. baculovirus/insect cells

Baculovirus-mediated protein expression in insect cells provides an additional means of preparing recombinant proteins (see Chapter 14 for details). BACULOVIRUSES are lytic, large (130 kb) double-stranded DNA viruses, of which Awiella virus is the most commonly used baculovirus for recombinant protein expression. The baculoviruses usually proliferate in insect cell lines derived from the fall armyworm moth Spodoptera frugiperda (Sf 9, Sf 21), and recombinant protein expression can be accomplished in the above cell lines as well as in cells derived from the cabbage looper Tii (HIghFiveTii). cells (HiGHFive) (Kost etal., 2005). Initially, the construction of recombinant baculoviruses involved the combination of baculoviral

Initially, the construction of recombinant baculoviruses involved cotransfection of insect cells with target genes having baculoviral D N A flanking sequences and baculoviral D N A, followed by screening for rare homologous recombinants. Recombinants are identified by screening for morphologically altered plaques, and additional rounds of plaque screening are often required to ensure that the recombinant virus is not contaminated with wild-type virus. This time-consuming and laborious method of preparing recombinant viruses has been replaced most of the time by site-specific transposition techniques (Bac^to-Bac or Bac uIoDirect, Invitrogen) and modified homologous recombination techniques (which are used to engineer baculoviruses containing a lethal mutation in orfi62 9) (flashBAC from Oxford Expression Technologies, or flashBAC from Oxford Expression Technologies, or flashBAC from Oxford Expression Technologies, or flashBAC from Oxford Expression Technologies). The technique was replaced by flashBAC from Oxford Expression Technologies, or BacMagic from EMD-No-Vague). Because of their 100% recombination efficiency, both methods omit the isolation of empty spots.After 1 or 2 rounds of recombinant virus amplification, researchers can quantify baculovirus concentrates using empty-spot analysis, the newer and more rapid real-time quantitative PCR assay, or an antibody-based assay (Hitchm an et al. , 2 0 02). Advances in recombinant baculovirus construction and quantification methods have dramatically reduced the time for expression using baculoviruses - to approximately 3 weeks, which includes the time taken to optimize expression.

The most commonly used baculovirus expression systems are the polH initiator and the plO initiator, both of which can be induced to produce high levels of expression late in the course of baculovirus infection (Ikonomou et al., 2003). 2003). At this stage, cell death begins , accompanied by the release of proteases that may lead to the degradation of recombinant proteins. To reduce the enzymatic degradation of recombinant proteins, promoters that are activated earlier in the cell lysis cycle, such as the basic prom oter, have been used (Ikonomou et al., 2003). The CA£A and 1)-○1 genes encode chitinase and a cathepsin protease, respectively, and it is also possible to minimize protein hydrolysis by constructing genetically defective versions of both (Monsma and Scott, 1997). 1997).

Baculovirus-mediated protein expression can often be used both to prepare recombinant proteins for cytoplasmic expression and to obtain recombinant proteins for secretory expression. Efficient secretory expression generally requires the involvement of signaling peptides. Both insect and mammalian signaling sequences can direct recombinant proteins into the secretory pathway of insect cells. Initially , insect cells were cultured in serum-containing media, which complicated the purification of secreted proteins. Recently, new media have been developed that allow the use of protein hydrolysates from animal or plant tissues instead of serum, thus greatly simplifying protein purification (Ikonomou et al., 2003). However, the high cost of this particular medium limits its use in large-scale production.

Insect cells can efficiently form disulfide bonds in recombinant proteins and also perform most of the post-translational modifications found in mammalian cells. However, most of the JV-linked glycans formed in insect cells are fucosylatedpaucimannose structures (Man3 GlcNAc2-N-A sn) (H arrison and Jarvis, 200 6 ; Jenkins e ta l. , 1 9 % ) . This discovery led to the recent construction of insect cell lines capable of producing glycoproteins containing complex N-linked glycans commonly found in mammalian cells (Harrison and Jarvis, 2006). A transgenic S f-9 insect cell line that expresses several glycosyltransferases has been commercialized (Mimiccelllin eInvitrogen), and the N-linked glycans it produces contain a double tentacle-like sialylated structure. Only a few papers have described the structure of O-linked glycans produced by insect cells (Chen et al. , 1991; Sugyiama eta l . , 1993; Thomsen et a l . 2004).

V. Nursing Animal Cells

Previously, mammalian expression methods were generally considered to be the least efficient way to express recombinant proteins. However, recent advances have dramatically increased the level of expression in mammalian cell lines (see Chapter 15 for details). For example, it has been reported that recombinant antibodies can be expressed at levels of several milligrams per liter using stably transfected Chinese hamster ovary (C H O ) cells (Figueroa et al., 2007; W urm, 2004). , 2007; W urm, 2004). Although a variety of cell lines and expression strategies have been tested, in this chapter we focus on transient transfection of human embryonic kidney (H E K 293) cells and stable transfection of C H O cells.

The H E K 293 cell line is derived from adenovirus-transformed human embryonic kidney cells. High transient transfection efficiencies (> 80 %) of H E K 293 cells were achieved using specific transfection reagents, such as cationic lipids, calcium ph o sp h ate, or polyethyleneimine (D u ro ch er et aL , 2002; Jo rd an etaL , 2002; D u ro ch er etaL , 2002; Jo rd an etaL , 2002; Jo rd an etaL , 2002; Jo rd an etaL , 2002; Jo rd an etaL , 2002; Jo rd an etaL , 2002; Jo rd an etaL , 2002; Jo rd an etaL , 2002). Jo rd an etaL , 1996). For large-scale transient transfection ( > i 0 0 mL), the use of calcium phosphate or polyethylsusceptible imide as transfection reagents is a more cost-effective choice than cationic liposomes (Baldi et a L , 2007). Transient transfection at the bioreactor level has been performed, although this scale is technically challenging for most laboratories (G irard etal., 2002). Transient transfection techniques are relatively simple and can be evaluated within 2 weeks for a given recombinant protein.

When large quantities of recombinant proteins are required, CHO cells are often chosen as the mammalian expression system. For example, most of the therapeutic antibodies currently on the market are produced using this cell line. The standard expression technique for stably transfected C H O cells involves transfection of D H F R-deficient CH◦ cells using vectors with a dihydrofolate reductase (D H FR) selection marker expression frame and a target gene expression frame (W urm, 2004). Dihydrofolate reductase reduces dihydrofolate to tetrahydrofolate, which is required for the de novo synthesis of purines, specific amino acids, and deoxythymidinelic acid. Methotrexate binds and inhibits DHFR and can be used as a selection reagent; only cells incorporating the DHFR selection marker expression frame will survive. Successive increases in methotrexate concentration result in massive amplification of the DHFR gene and its linked target gene. After at least one round of methotrexate screening, subcloning of the stably transfected cell population is achieved by cloning the stably transfected cells into multiwell plates using the limited dilution method. As is often the case, only a small fraction of the screened subclones are able to express the recombinant gene at a high level because the expression frames are integrated into transcriptionally inactive heterochromatin regions in most clones. Unfortunately, the whole selection and screening process takes at least 2 to 3 months, which is the main drawback of the CHO expression method. However, current high-throughput techniques based on flow and automation technologies have greatly simplified the rapid screening and selection of highly expressed clones (Browne and Al-Rubeai, 2007). Another advance has been the use of specific cis-acting D N A elements (e x a c tin g DNA elements) located on either side of the expression frame of recombinant genes, which can make the integration site transcriptionally active (Kwaks and O tte, 2006). Unfortunately, most of these D N A elements are owned by companies, and laboratories must obtain a license from the company to use them. In addition, even with the advances described above, the time required to obtain highly expressed C H O clones has not changed much.

Mammalian expression systems have been used primarily for the preparation of secreted recombinant proteins rather than for the preparation of intracellularly expressed proteins. Serum-free medium has been developed for C H O and HEK293 cell lines, which makes purification of secreted recombinant proteins simple. However, this medium is expensive, making large-scale production very expensive.

Mammalian cells have the most excellent protein folding and disulfide bond forming ability compared to hosts of other expression systems. The structure of N-linked glycans and 0-linked glycans produced by mammalian cells is highly variable, depending not only on the protein but also on the type of mammalian cell that serves as the expression host (Jenkins et al., 1996). In addition, cell culture conditions such as nutrient composition, p H , temperature, oxygen levels, and ammonia concentration are capable of significantly affecting glycosylation (Butler, 2006). JV-linked glycosylated sugars will be oligomannans (○ lig ○ mannose), heteroglycans, and other complex glycan structures, but all will contain - a Man3 GlcNac2 core (Bhatia and Mukhopadhyay, 1998). Oligomannans have 2 to 6 additional mannose residues that can be phosphorylated or sulfated. The most common complex glycan structure has 2 to 4 Gal,4-GlcNac2 groups attached to the mannose, which are capable of forming two, three and four tentacle-like branching structures. The branching structure ends in sialic acid, and fucose can also be attached to the branch. Heteroglycans are characterized by both oligomannose and complex glycan structures ^ O-linked glycosylation structures can be classified into eight categories based on their core structure: 0 GalN Ac-type glycosylation, O GlcN A-type glycosylation, O Fucosylation, O GlcN A-type glycosylation, O Fucosylation, O GlcN A-type glycosylation, O GlcN A-type glycosylation, O GlcN A-type glycosylation, O GlcN A-type glycosylation, O GlcN A-type glycosylation, and O Fucosylation. Ofucosylation, Om annosyiation, O glucosylation, phosphoglycosylation, O glucosaminoglycan type glycosylation, and O Glycosaminoglycan type glycosylation (○ - -glyc ○ saminaminoglycan type glycosylation). O-glyc 〇 saminoglycan-type glycosyiation) and collagen-type glycosylation (Peter-Katalinic, 2005).

VI Protein characterization

When selecting an expression system, it is easy to research the literature to confirm whether the recombinant protein has been previously expressed and to evaluate published expression strategies. When borrowing from the literature, it is important to consider whether the purpose for which the recombinant protein is used in the literature is similar or compatible with your intended application. Reports expressing or detailing homologous proteins are also useful when literature information is lacking. There has been a dramatic increase in the number of proteins expressed over the past decade, thanks in part to the determination of multiple genome sequences, the development of high-throughput expression methods, and the large-scale protein structure initiative (P S I ). This trend is likely to continue, which means that eventually the large amount of data from previous generations will make the selection of expression systems much simpler and easier.

1. Protein characterization: E. coli and codon usage

-In general, in the selection of expression hosts for recombinant proteins, proteins of prokaryotic origin should only be expressed in E. coli and not in eukaryotic expression systems, because usually the post-translational modification capabilities and improved folding abilities of eukaryotes are not necessary, or even desired, for prokaryotic proteins. The situation is different for eukaryotic proteins, as there are numerous examples of eukaryotic proteins being successfully expressed in E. coli (Sahdev et al., 2008). A very important consideration when expressing eukaryotic proteins using a prokaryotic system is that, like all organisms, E. coli has a bias for codon usage, and its t R N A abundance reflects this -bias (Gustafsson et al., 2004; Marin, 2008). Expression of eukaryotic proteins containing several E. coli rare codons is inefficiently limited by the corresponding t R N A abundance. The shortage of tR N A leads to translational frame shifting, amino acid misincorporation and premature termination of translation. This problem is particularly evident when rare codons are concentrated at the N-terminus (Kane, 1995). However, this problem can be avoided by synthesizing codon-optimized genes or by using commercially engineered strains (e.g., strain R osetta, E M D --N o v a g e n ) with increased abundance of rare codons t R N A . In most cases, codon bias also needs to be corrected if the recombinant gene is expressed in a distantly related host organism.

2. Protein characterization/cytoplasmic proteins

For cytoplasmic proteins, the choice of the best expression system depends on the size of the protein and the number of disulfide bonds in the molecule. For proteins with a molecular mass of 10-50 kD a and very few disulfide bonds, E. coli is a good choice for soluble expression of proteins (D y s o n e ta L , 2004). For larger proteins or proteins with many disulfide bonds, a baculovirus or fermentation system is usually preferred if soluble expression is desired. Proteins with few or no disulfide bonds below 10 kDa have been successfully expressed in E. coli by fusing soluble tags (Esposito and Chatterjee, 2006). Alternatively, it is also possible to express these small proteins in Picrosporum for secretion (Daly and H earn, 2005). However, this pathway must be carefully monitored as unintentional glycosylation can occur when proteins normally present in the cytosol are forced into the secretory pathway. This can be achieved by checking the sequence to confirm that it does not contain a consistent N-linked glycosylation site. Unfortunately, there is no conserved sequence for N-linked glycosylation, so secreted recombinant proteins must be analyzed to ensure that they do not contain N-linked glycans.

3. Protein characterization: Secreted proteins

All expression hosts can be used to produce secreted proteins. However, as mentioned previously, E. coli lacks most of the post-translational modification functions found in eukaryotes. Therefore, E. coli may not be suitable for the expression of secreted eukaryotic proteins, although this also depends greatly on the downstream application.

4. Protein characterization/membrane proteins

Membrane proteins are very challenging to express in large quantities. In some cases, it is sufficient to express only the soluble, hydrophilic portion, where the transmembrane domains can be removed and the soluble portion expressed. For the expression of intact membrane proteins, there are no clear guidelines on how to choose the best expression system (Sarramegna et al., 2003). However , for most eukaryotic membrane proteins, the E. coli expression system is usually not a good choice due to limitations in folding and post-translational modification capabilities. In contrast, researchers have reported some success in achieving expression of G protein-coupled receptors using baculovirus and yeast expression systems.

5. Protein characterization/toxic proteins

Recombinant proteins that are toxic to the expression host can pose a challenge to expression, but this can usually be overcome. If the recombinant protein is toxic, it is often useful to determine if the toxicity is host cell specific. If it is, then expression in a more compatible expression host may be an option. Another option is to use tightly regulated, inducible expression systems such as those applied in E. coli and Pichia yeast. For example , several precisely regulated inducible E. coli expression systems have been developed (Saida, 2007). In these systems, the expression of recombinant genes is regulated by inducible promoters, transcriptional terminators, control of plasmid copy number, or modification of the coding sequence of the recombinant gene. In the available Picot yeast system, the A O X l promoter is tightly regulated by a combination of inducible mechanisms as well as deterrence/de-deterrence approaches (Daly and H eam , 2005). In addition, several studies have shown that baculovirus/insect expression systems can be used to express toxic proteins (Aguiar et al., 2:006; Korth and Levings, 1993). Finally , the simplest option for mammalian expression systems is transient expression. Several inducible expression systems in C H O cells require considerable time to complete the necessary cellular engineering modifications, and it is difficult to utilize these expression systems to obtain the tightly regulated gene expression that is required to prevent cell death (Rossi and Blau, 1998).

VII. Applications of recombinant proteins

When used for structural studies, the expression of recombinant proteins requires correct protein folding, formation of correct disulfide bonds, and homogeneous recombinant products. The intrinsic ability of each expression system to fold proteins and fo

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