Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Fundamentals of Molecular Biotechnology
Production of Recombinant Proteins Using Eukaryotic Systems

Prokaryotic expression systems are typically used to produce heterologous recombinant Proteins from cloned eukaryotic complementary DNA (cDNA). However, in some cases, eukaryotic proteins synthesized in Bacteria prove to be unstable or biologically inactive. Furthermore, no matter how rigorous the purification process, the final product may still be contaminated with toxic substances or pyrogens—substances that induce fever in humans and animals. To overcome these limitations, eukaryotic expression systems were developed for The production of recombinant proteins intended for medical Applications. Such proteins must be identical to their natural counterparts in biochemical, physical, and functional properties. The inability of prokaryotes to synthesize authentic protein variants is primarily due to their lack of adequate mechanisms for introducing specific post-translational modifications.

Proteins undergo the following post-translational modifications in Eukaryotic Cells.

✵ Formation of Disulfide Bonds, a reaction catalyzed by the enzyme protein disulfide isomerase. Improperly folded proteins turn out to be unstable and inactive.

✵ Proteolytic Cleavage of a precursor, involving the removal of a specific segment of the polypeptide chain to yield a functionally active protein.

✵ Glycosylation: a major modification that imparts stability and, in some cases, specialized properties to proteins. The most common glycosylation reactions involve the attachment of a specific sugar residue either to Serine or Threonine (O-glycosylation) or to asparagine (N-glycosylation).

✵ Amino acid modifications within the Cell/13.html">Protein Structure: phosphorylation, Acetylation, acylation, gamma-carboxylation, sulfation, myristoylation, and palmitoylation.

Among all these modifications, prokaryotic host cells are least capable of properly carrying out glycosylation and specific amino acid modifications in a heterologous protein. However, no single eukaryotic system can perform all post-translational modifications simultaneously for every potential heterologous protein. Therefore, to obtain a protein with the complete set of specific modifications, various eukaryotic expression systems must be tested to identify one that reproduces a biologically authentic product.

Eukaryotic expression vectors share a structural layout similar to that of their prokaryotic counterparts (Fig. 7.1) and must contain:

✵ a eukaryotic selectable marker

✵ a eukaryotic promoter

✵ appropriate Eukaryotic METABOLISM/31.html">Transcription and Translation termination sites

✵ an mRNA polyadenylation signal.

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Fig. 7.1. General structure of a eukaryotic expression vector. Its main elements include: a eukaryotic transcripton comprising a promoter (p), a cloning site (CS), and termination and polyadenylation signals (t); a eukaryotic selectable marker (SM); an origin of Replication functional in eukaryotic cells (ori^euk); an origin of replication functional in E. coli (ori^E); and an E. coli selectable marker (Amp^r).

If the vector is a plasmid that replicates independently of the chromosome, it must contain an origin of replication functional in the host cell. Conversely, if the vector is designed to integrate into the host chromosomal DNA, it must carry a sequence complementary to a specific region of the host chromosomal DNA (chromosomal integration site) to ensure recombination. Because many recombinant DNA Procedures are technically more challenging to perform in eukaryotic cells than in prokaryotic ones, most eukaryotic vectors are designed as shuttle vectors. In other words, these vectors carry Two Types of initiation sites and two types of selectable marker genes—one set functioning in Escherichia coli and the other in eukaryotic host cells. Such vector expression systems have been developed for Yeast, insect, and mammalian cells.

The Introduction of DNA into bacterial and yeast cells is referred to as transformation. In microbiology, this term describes heritable changes resulting from the uptake (acquisition) of exogenous (foreign) DNA. In contrast, when applied to animal cells, transformation denotes an alteration in their growth pattern in culture caused by The conversion of normal cells into Cancer cells. To avoid terminological confusion, the term transfection was chosen to designate heritable changes in animal cells following the introduction of exogenous DNA.

Three main Methods are commonly used for yeast transformation. In the first approach, exogenous DNA is added to yeast cells whose cell walls have been removed chemically or enzymatically (protoplasts) (1). Alternatively, cells are treated with lithium acetate prior to The addition of foreign DNA (2) or subjected to electroporation (3). Transfection of Animal Cell Cultures is performed by incubating cells with DNA precipitated using calcium phosphate or DEAE-dextran (1), or by electroporation in the presence of purified transfecting DNA (2). As mentioned in Chapter 4, electroporation involves exposing cells to brief, high-voltage electrical pulses, which creates temporary Pores in the outer membrane or Cell wall through which DNA can enter The Cell. In some eukaryotic systems, Viruses are used to deliver DNA into recipient cells.