Molecular Biotechnology. Bernard R. Glick

as inactive precursor polypeptides that must be cleaved by proteases at specific sites to produce the active form of the protein. For example, the small peptide hormone insulin is produced in animal pancreatic cells as a single polypeptide, preproinsulin, that is cleaved to produce two shorter peptides that are joined by disulfide bonds (Fig. 3.22). Production of inactive preproinsulin ensures that the peptide is not active in the pancreatic cells that produce it, but upon secretion, cleaved mature insulin can act on other cells. Similarly, the digestive enzyme trypsinogen, which degrades proteins, is produced as an inactive polypeptide to avoiding digestion of components of the producing cell. Upon secretion into the small intestines, trypsinogen is cleaved by an enteropeptidase to yield the active enzyme trypsin.

      Figure 3.22 Cleavage of inactive preproinsulin to yield active mature insulin. Proteases remove the leader peptide (L) and an internal peptide (C), yielding a peptide that consists of chains A and B.

      Similar to prokaryotes, proper folding of proteins in eukaryotic cells requires the assistance of chaperones. In the endoplasmic reticulum, the chaperones BiP and calnexin bind nascent polypeptides, and protein disulfide isomerases catalyze the formation of disulfide bonds between cysteines. Proper folding is important, not only for the protein to attain a configuration for optimal activity, but also to protect regions of the protein that would otherwise be recognized by proteases that can destroy the protein. Quality control systems ensure that only correctly folded proteins are released from the endoplasmic reticulum and transported within vesicles to the Golgi apparatus for further processing. Proteins intended for secretion from the cell are subsequently transported to the cell membrane within specific transport vesicles and released by exocytosis.

      The addition of specific sugars (glycosylation) to certain amino acids is a major posttranslational modification that provides stability and distinctive binding properties to a protein. Proper protein glycosylation is important because it contributes to protein conformation by influencing protein folding; can target a protein to a particular location, for example, through interaction with a specific receptor molecule; or can increase protein stability by protecting it from proteases. In the cell, oligosaccharides are attached to newly synthesized proteins in the endoplasmic reticulum and in the Golgi apparatus by specific enzymes known as glycosylases and glycosyltransferases. Different tissues may differentially glycosylate the same protein, thereby increasing protein heterogeneity. Because different sugar modifications can alter the properties of a protein, this presents opportunities for protein engineering to improve the efficacy or to alter the activity of a protein. About 50% of all human proteins are glycosylated. Human therapeutic proteins that require glycosylation for optimal activity include antibodies, blood factors, some interferons, and some hormones.

      The most common glycosylations entail the attachment of specific sugars to the hydroxyl group of either serine or threonine (O-linked glycosylation) (Fig. 3.23) and to the amide group of asparagine (N-linked glycosylation) (Fig. 3.24). The initial core sugar groups that are added to these amino acid acceptor sites tend to be similar among eukaryotes, although the subsequent elaborations among yeasts, insects, and mammals are quite diverse, especially for N-linked glycosylation. Other amino acid modifications include phosphorylation, acetylation, sulfation, acylation, γ-carboxylation, and the addition of C14 and C16 fatty acids (i.e., myristoylation [or myristylation] and palmitoylation [or palmitylation], respectively).

      Figure 3.23 Examples of some O-linked oligosaccharides in yeasts (A), insects (B), and mammals (C). O-linked oligosaccharides have a number of arrangements with different combinations of sugars. Some of the more prevalent forms are shown here. S, serine; T, threonine; red circles, mannose; dark-blue squares, N-acetylglucosamine; light-blue squares, N-acetylgalactosamine; green squares, galac- tose; orange squares, sialic acid.

      Figure 3.24 Examples of some N-linked oligosaccharides in yeasts (A), insects (B), and mammals (C). All N-linked glycosylations in eukaryotes start with the same initial group, which is subsequently trimmed and then elaborated in diverse ways within and among species. Some yeast sites have 15 or fewer mannose units (core series), and others have more (outer-chain family). In S. cerevisiae, the chains frequently have 50 or more mannose units. An asparagine (N) next to any amino acid (X) followed by either threonine (T) or serine (S) can be targeted for glycosylation. Red circles, mannose; dark blue squares, N-acetylglucosamine; yellow triangles, glucose; green squares, galactose; orange squares, sialic acid; maroon triangle, fucose.

      Unfortunately, there is no universally effective eukaryotic host cell that performs the correct modifications on every protein. In some cases, a host cell may add unusual sugars to either authentic or spurious amino acid sites and, consequently, create an extremely antigenic protein or possibly one that lacks its proper function. Even though a recombinant protein may fall short of the stringent properties that are required for a therapeutic agent, it may still be useful for either research or industrial purposes. Different eukaryotic expression systems must be tested to determine which one synthesizes the largest amount of a functional recombinant protein. The choice of an expression system depends primarily on the quality of the recombinant protein that is produced, but the yield of product, ease of use, and cost of production and purification are also important considerations.

General Features of Eukaryotic Expression Systems

      The basic requirements for expression of a target protein in a eukaryotic host are similar to those required in prokaryotes. Vectors into which the target gene is cloned for delivery into the host cell can be specialized plasmids designed to be maintained in the eukaryotic host, such as the yeast 2μm plasmid; host-specific viruses, such as the insect baculovirus; or artificial chromosomes, such as the yeast artificial chromosome (YAC). The vector must have a eukaryotic promoter that drives the transcription of the cloned gene of interest, eukaryotic transcriptional and translational stop signals, a sequence that enables polyadenylation of the mRNA, and a selectable eukaryotic marker gene (Fig. 3.25). Because recombinant DNA procedures are technically more difficult to carry out with eukaryotic cells, they are typically carried out in prokaryotic cells before transferring the vectors to the eukaryotic host for protein expression. Therefore, most eukaryotic vectors are shuttle vectors with two origins of replication and two separate selectable marker genes; one set of these genes functions in the bacterium E. coli, and the other set functions in the eukaryotic host cell. If a eukaryotic expression vector is to be used as a plasmid (i.e., as extrachromosomal replicating DNA), then it must also have a eukaryotic origin of replication. Alternatively, if the vector is designed for stable integration into the host chromosomal DNA, then it must have a sequence that is complementary to a segment of host chromosomal DNA to facilitate insertion into a chromosomal site.

      Figure 3.25 Generalized eukaryotic expression vector. The major features of a eukaryotic expression vector are a eukaryotic transcription unit with a promoter (p), a multiple cloning site (MCS) in which to insert a target gene, and a DNA segment with transcription termination and polyadenylation signals (t); a eukaryotic selectable marker (ESM) gene; an origin of replication that functions in the eukaryotic host cell (orieuk); an origin of replication that functions in E. coli (oriE); and an E. coli selectable marker (e.g., Ampr) gene.

      The introduction of DNA into bacterial or fungal cells is called transformation. In these systems, the term describes an inherited change due to the acquisition of exogenous (foreign) DNA. However, in animal cells, transformation refers to changes in the growth properties of cells in culture after