BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Recombinant Gene Expression Systems

When utilizing recombinant Gene Expression systems, the following key molecular and biological features are taken into account:

- type of promoter and METABOLISM/31.html">Transcription terminator;

- strength of mRNA-ribosome binding;

- copy number of the cloned gene and its localization (within a plasmid or the host Cell chromosome);

- localization of the synthesized product;

- Translation efficiency in the host Organism;

- product stability within the host cell.

Bacterial Cells, Yeast cells, Introduction/5.html">Eukaryotic Cell Cultures, cell-free protein-synthesizing systems, and continuous-flow systems are employed as RECOMBINANT PROTEIN EXPRESSION systems.

E. coli Bacteria synthesize a wide range of Enzymes (Hydrolases) and possess highly efficient mechanisms for enzyme folding and secretion. However, bacterial cells lack systems for post-translational protein modifications; consequently, biologically fully functional eukaryotic Proteins cannot be produced in them. More than 500 post-translational protein modifications are known, the primary ones being:

- formation of Disulfide Bonds;

- partial proteolysis – removal of a peptide from the N-terminus of the protein molecule to yield an active protein;

- glycosylation – attachment of a specific carbohydrate residue;

- modification of Amino Acids within the protein composition (phosphorylation, Acetylation, carboxylation, sulfation).

Prokaryotes are incapable of carrying out glycosylation and the modification of specific amino acids. Therefore, Eukaryotic cells are predominantly used for the synthesis of human proteins.

Yeast cells of Saccharomyces cerevisiae are used for the synthesis of human, animal, and plant proteins. Over 300 recombinant proteins are produced using methylotrophic Yeasts such as Pichia pastoris, which utilize methanol as a carbon source. Methylotrophic yeasts were first employed for the synthesis of Human and Animal proteins in 1984.

Cell cultures used for recombinant Protein Synthesis include:

- Chinese hamster Ovary cells (CHO cell line);

- mouse myeloma cells (SP 210 cell line);

- mouse Spleen cells (MEL cell line);

- African green monkey cells (COS cell line);

- insect cells infected with Baculoviruses.

A distinctive feature of insect cells is their ability to perform post-translational protein modifications, although these differ somewhat from modifications occurring in mammalian cells. The most commonly used cell lines are IPLB-Sf-21-AE from Spodoptera frugiperda and BTI-TN-5B1-4 from Trichoplusia ni.

A comparison of bacteria, yeast cells, insect cells, mammalian cells, human cells, and Transgenic Plants and animals as expression systems for recombinant genes is presented in Table 1.2.

Class="center">Table 1.2

Capabilities for the Production of Human proteins based on recombinant

DNA technology

Cells with compromised outer membrane permeability

In terms of their MECHANISM OF ACTION and properties, these cells occupy an intermediate position between living cell translation systems and cell-free protein-synthesizing systems. To obtain cells with permeabilized membranes, animal cells are cultured and treated with the detergent digitonin. Semi-permeable membrane cells are used to investigate protein secretion processes.

Cell-free protein-synthesizing systems are used to study the mechanisms of cloned GENE EXPRESSION IN vitro and to produce recombinant proteins. The advantage of these systems is the accessibility of their individual components for experimental manipulation. Cell-free systems make it possible to study The Influence of various exogenous factors (pH, activators, inhibitors) on their functioning.

There are 2 types of cell-free protein-synthesizing systems:

1) prokaryotic systems (based on E. coli cell extracts) containing the following components: Ribosomes, translation factors, mRNA, tRNA, amino acids, GTP, K⁺/NH₄⁺, Mg²⁺/Ca²⁺, buffers;

2) eukaryotic systems derived from rabbit reticulocytes, wheat germ (suitable for long-term storage), and somatic cells of various origins.

Cell-free systems have a short lifespan (40-60 min) and provide a low yield of synthesized protein (1 µg / 1 mL of extract), with only 1-2 polypeptide molecules synthesized per mRNA molecule.

Continuous-flow systems

In 1988, the Russian scientist A.S. Spirin developed an efficient continuous-flow protein-synthesizing system. Bacterial or eukaryotic cell extracts are placed in a chamber enclosed on both sides by semi-permeable membranes. The pore size allows low-molecular-weight substances and small proteins to pass through the membranes. The Contents of the chamber containing The Cell-free translation components are incubated at a standard Temperature. Meanwhile, the ingredients required for Protein Biosynthesis (amino acids, ATP, GTP) are continuously fed into one side of this Reactor chamber at a rate of 1 mL/h, while the synthesized protein products exit from the other side.

In continuous-flow systems, protein biosynthesis can proceed uninterrupted for several hours. Translation efficiency is quite high: hundreds of copies of polypeptide chains are synthesized per single mRNA molecule. The total yield of recombinant protein reaches 200 µg or more per 1 mL of extract.

Continuous-flow protein-synthesizing systems have made it possible to carry out The biosynthesis of recombinant proteins in preparative quantities in vitro. Such systems allow the synthesis of Polypeptides that are subject to rapid intracellular degradation by proteinases, or proteins with cytotoxic activity. Preparative amounts of functionally active human interleukin-6 can be obtained using continuous-flow systems.