2.1 Overview: Protein pharmaceuticals
Biotechnology have been used to make bioactive products for medical or research purposes for a long time. The biotechnology inductry have been hugels succesful in producing simpler, smaller molecules, such as antibiotics, but the mass production of human proteins (or even smaller peptides) proved to be significantly more complicated. Many hormones are in fact small peptides (calcitonin, oxytocin, vasopressin) and can be synthesized using the method developed by Bruce Merrifield in the sixties. Chemical peptide synthesis is very efficient (over 99% per bond), but that means that for even a short 50-amino acid peptide the overall yield will be 60.5%. Traditionally, human or animal proteins intended for medical purposes are isolated from natural sources, using complicated protein purification techniques. Human and animal tissues, including blood can be sued as natural source for several important proteins. 8-9% of blood consists of 10000 different proteins, although the bulk of this is really made up from about 20 major blood proteins. Blood clotting factors for the treatment of coagulation diseases were for a long time isolated from human blood using Cohn fractionation developed in 1946. A great advantage of proteins isolated from human blood is the species-identity, which prevents the mounting of immune response against the protein product, and the human proteins also carry all posttranslational modifications necessary for their biological activity. At the same time, there never is a sufficient supply of human blood, and any protein pharmaceuticals isolated from human or animal tissues may contain traces of infectious agents (known or unknown), and hazardous contaminants incompletely removed during purification. Recombinant DNA technology provides the means to produce human protein pharmaceuticals in simple organisms, utilizing well-established fermentation technologies. There are four general approaches to produce recombinant proteins:
1. Expression in cell-free systems (in vitro) 2. Expression in isolated cells (cell cultures) 3. Expression in transgenic plants/animals 4. Gene therapy in humans
During this lecture we will discuss the first two systems, comparing the advantages and disadvantages of the in vitro and cell culture systems, and their applications.
2.2 Cell-free systems: In vitro transcription and translation
Cell-free systems are exceptionally useful for research purposes, but the low yields excludes their utilization for the mass production of proteins. There advantages over in vivo gene expression are:
Identification number:
TÁMOP-4.1.2-08/1/A-2009-0011 17
• When the protein is: toxic to the host cell, insoluble or forms inclusion bodies, degraded rapidly by intracellular proteases
• Speed and directness of all procedures
• Absence of constraints from a living cell
• Pure product
Disadvantages over in vivo gene expression are:
• Lack of cellular membranes
• Lack of post translational modifications
Eukaryotic transcription in vivo is based on the specific interaction of several proteins, the DNA template and the newly produced RNA. The majority of the proteins are involved in regulation and posttrancriptional processing. Since the majority of these functions are not needed in an in vitro system, we can utilized the much simpler transcriptional system of the bacteriophages. For this we need the phage RNA polymerase, nucleotides and the appropriate buffer.
On the other hand, eukaryotic translation requires ribosomes, tRNAs, amino acids, template RNA and a host of other proteins, not all well characterized. Therefore, in vitro translation systems are always provided as crude extract of cells.
Figure 2.1. Linearization of template
2.3 Recombinant protein expression in isolated cells (cell culture)
The first step of recombinant protein expression is identification and cloning of its gene. This process is the subject of recombinant DNA technology, and will not be discussed in this lecture. A critical decision point is determining which expression system to use: in vitro or in vivo, and if in vivo, which species to choose. Culturing and protein purification conditions are based on standard approaches, but always require empirical optimization. Initially prokaryotes were used to produce recombinant proteins - however, for larger proteins, or proteins with extensive posttranslational modifications eukaryotic sytems are better suited.
18 The project is funded by the European Union and co-financed by the European Social Fund.
2.4 Non-prokaryotic expression systems 2.4.1 Cloning in Pichia pastoris
This system uses a special plasmid that works both in E. coli and yeast.
Once the gene of interest is inserted into this plasmid, it must be linearized, then yeast cells are transfected with linear plasmid. Following transfection double cross-over recombination event occurs to cause the gene of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be. From now on the gene of interest is under control of the powerful AOX promoter.
2.4.2 Baculovirus mediated protein expression in insect cells
This system uses the Autographica californica multiple nuclear polyhedrosis virus (Baculovirus), which commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae). The system uses super-strong promoter from the polyhedrin coat protein to enhance expression of proteins while virus resides inside the insect cell - protein is not required for infection or viral life cycle. Secreted proteins better expressed by stably transfected insect cell lines, from the ie-1 promoter (infection interferes with secretory pathways).
Figure 2.2. Baculovirus/insect expression system 2.4.3 Mammalian expression systems
In these systems the gene is initially cloned into plasmid, and propagated in bacterial cells, then the mammalian cells are transformed by electroporation (with linear plasmid) and the gene integrates (1 or more times) into random locations within different chromosomes. The cells are typically derived from the Chinese Hamster Ovary (CHO) cell line. Often, multiple rounds of growth and selection using methotrexate to select for those cells with highest expression &
integration of DHFR and the gene of interest.
Identification number:
TÁMOP-4.1.2-08/1/A-2009-0011 19
Figure 2.3. Methotrexate (MTX) selection
2.5 Purification of recombinant proteins
Protein purification protocols produce proteins with variable purity - as always, therapeutical proteins have to be extremely pure. Steps of the purification strategies are based on different physicochemical properties of the proteins, such as size, charge, side chains, hydrophobicity. This requires several purification steps, lengthy optimization for ech protein, and sensitive and specific detection of the protein at each step. For recombinant proteins on the other hand, it is possible to attach a short protein sequence, a so called tag to the N or C terminal of the protein using recombinant DNA technology. The tags are designed to bind smaller molecules or metal ions with high affinity and specificity, and can greatly shorten and standardize the purification of a wide variety of proteins.
Figure 2.4. Conventional purufucation strategy
20 The project is funded by the European Union and co-financed by the European Social Fund.