Production of tpa using eukaryotic n prokaryotic cells - Essay Example

The Production Of tPA Affiliation Production of drugs has eased treatment of various ailments and their symptoms. Through testing, packaging, marketing and promotion the drugs and medicines manufacturing industry is operational a on full scale basis. Therefore treatment through the use of drugs is more effective and efficient. Due to their high demand, drugs are scarce leading to sky rocketing of prices thus raising questions concerning the cost of producing drugs. Production of newly discovered drugs is expensive and requires huge sums of money during packaging and marketing.

During the course of production, safety precautions have to be strictly adhered to, as the drugs are poisons in nature and could cause severe body harm if misused. A method for producing tissue plasminogen activator (t-PA) in eukaryotic host cells is disclosed. Enhanced levels of t-PA production are obtained by co-amplification of the t-PA gene through treatment of cultures transformed with mutant or wild type DHFR with methotrexate. A cell culture comprising methotrexate (MTX) sensitive recombinant host cells transformed with an expression vector comprising a first DNA sequence encoding a dihydrofolate reductase (DHFR) protein with a low binding affinity for MTX, and a second DNA sequence encoding human tissue plasminogen activator (tPA), tPA encoded by said second DNA sequence, and an effective amplifying concentration of MTX.

The invention herein relates to the production of human tissue plasminogen activator (tPA) in a transformant host cell culture. More specifically, the invention relates to vectors, cells, and methods of producing tPA in conjunction with expression of the sequences for coding for dihydrofolate reductase (DHFR) protein in such cells.In selecting a preferred host cell for transfection by the vectors of the invention, it is appropriate to select the host according to the type of DHFR protein employed.

Another example that describes use of CHO cells as host cells, and expression vectors which include the SV40 origin of replication as a promoter. However, it would be well within the skill of the art to use analogous techniques to construct expression vectors for expression of desired protein sequences in alternative eukaryotic host cell cultures. If cells without formidable cell wall barriers are used as host cells, transfection is carried out by the calcium phosphate precipitation method as described by Graham and Van der Eb, Virology, 52:546 (1978).

However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used.Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and relegated in the form desired to form the plasmids required. Cleavage is performed by treating with restriction enzyme (or enyzmes) in suitable buffer.. After incubations, protein is removed by extraction with phenol and chloroform, and the nucleic acid is recovered from the aqueous fraction by precipitation with ethanol.

The following examples are intended to illustrate but not to limit the invention. In the examples here, a CHO cell line suitable for the type of DHFR protein coding sequence to be introduced was employed as a host cell culture in each case. However, other eukaryotic and prokaryotic cells are suitable for the method of the invention as well. Temperature shift Low culture temperature has been shown to reduce the growth rate, increase culture longevity and specific productivity in CHO cells expressing wide range of recombinant proteinsIn this laboratory reducing the temperature of suspension CHO-K1 cells from 37 to 31°C during the latter stages of exponential growth resulted in an immediate cessation of proliferation.

Cultures grown at 31°C achieved peak viable cell numbers that were almost 20% lower than in cells cultured for another 24 h at 37°C). A shift in the proportion of cells from the S to the G1 phase of the cell cycle has been observed at reduced temperatures which results in a state close to growth arrest and has been shown to improve productivity is still poorly understood although recent studies have demonstrated that reduced culture temperature invokes a coordinated response involving the cell cycle, transcription and translational machinery, and the arrangement of the.

Low culture temperature results in reduced metabolism and shear sensitivity which leads to delayed initiation of apoptosis and helps in extending the duration of the stationary/production phase. Reduced culture temperature has also been shown to increase levels of recombinant mRNA, either due to enhanced transcription of the recombinant gene of interest or increased mRNA stabilityEffect of reduced temperature on cell cycle and productivity in CHOCurrently there is very little known regarding the mammalian cell response to reduced culture temperature at the molecular level.

Finding alternative compounds that can be used efficiently as carbon and energy sources with lower uptake rates is important because slow consumption of nutrients can lead to reduced/arrested cell growth. These changes are associated with improved culture longevity and overall productivity.The exponential increase in the demand for therapeutic protein products is the major driving force to improve current production systems. Proliferation and regulation of the cell improves cell characteristics and production in CHO systems.

Comparisons of temperature, chemical and nutritional approaches, cell engineering, growth regulation through low temperature cultivation has been proven to be more reliable and efficient. Reduced temperature cultivation improves the cells’ overall productivity. Although the effects of reduced temperature on productivity can be impressive, it should be noted that it still varies among clones, cell lines and recombinant protein products and is still poorly understood; however, further research in this field will understand the implication of cold on cells with the aim of further.

ReferencesAltamirano C, Paredes C, Cairo JJ, Godia F (2000) Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine. Biotechnol Prog.Baik, J.Y, Lee M.S, An SR, Yoon SK, Joo EJ, Kim YH, Park HW, Lee GM (2006) Initial transcriptome and proteome analyses of low culture temperature-induced expression in CHO cells producing erythropoietin. Biotechnol Bioeng.