EXPRESSION SYSTEMS.ppt
- 1. HOSTS FOR GENE CLONING Dr. Esther Shoba R Assistant Professor Department of Life Sciences Kristu Jayanti College
- 2. INTRODUCTION • The development of genetic engineering and cloning has opened many possibilities of expression and isolation of heterologous proteins for research purposes. • Large scale applications such as enzyme, antibody or vaccine production, the amount of protein required is considerably high. • In such cases the system in which the protein is expressed must be easy to culture and maintain, grow rapidly, and produce large amounts of protein. • These requirements led to the discovery of protein expression systems. The various protein expression systems are bacteria, yeast, insect or mammalian systems.
- 3. • The following factors determine the type of expression system used to produce recombinant proteins: • time spent in expressing the protein • ease of handling the expression system • amount of protein needed • mass of the protein • type of post-translational modifications, number of disulfide bonds • destination of the expressed protein
- 4. • The process of expressing a recombinant protein in an expression system requires the following information/components. • identification of the gene that encodes the protein of interest • generation of cDNA from the respective mRNA • selection of suitable expression vector to insert the gene sequence • selection of suitable system that can express the vector • appropriate screening and scaling up methods
- 6. Bacterial protein expression systems – Escherichia coli • Bacteria act as rapid and simple systems of expressing recombinant proteins due to the short doubling time. • The media required to culture them are not expensive and the methods adapted to scale-up bioproduction are straightforward. • • The most widely used host system is E. coli since there is ample knowledge about its genetics, genome sequence and physiology. • The genetic manipulation is easy and it also grows to high densities and is suitable for large-scale fermentations. • However, the cell wall of E. coli contains toxic pyrogens and the expressed proteins may have to be extensively tested before use.
- 8. • Features • low cost culture methods • flexible system – can carry plasmids with multiple promoters, tags and restriction sites • easy to scale up and produce higher yield of protein
- 9. DH5 ALPHA • DH5 alpha is the most frequently used E. coli strain for routine cloning applications. In addition to supporting blue/white screening recA1 and endA1 mutations in DH5a increase insert stability and improve the quality of plasmid DNA prepared from minipreps. • Application: • Highest transformation efficiency • General cloning • Blue-white selection • Plasmid isolation
- 10. • The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue in order to disable the activity of the recombinases and inactivate homologous recombination. • The endA1 mutation inactivates an intracellular endonuclease to prevent it from degrading the inserted plasmid. • High insert stability due to recA1 mutation • High yield and quality of DNA due to endA mutation
- 11. BL 21 HOST CELLS • BL21 Competent E. coli is a widely used non- T7 expression E. coli strain and is suitable for transformation and protein expression. • Ideal for Plac, Ptacexpression vectors • Protease deficient
- 12. BL21 DE 3pLysS • BL21(DE3)pLysS is a derivative of BL21 that has the T7 RNA polymerase gene under the control of the lacUV5 promoter. • This arrangement is on a phage genome, called DE3. • DE3 is inserted into the chromosome of BL21 to make BL21(DE3). • pLysS is a plasmid that contains the T7 lysozyme gene (LysS). • The T7 lysozyme binds to T7 RNA polymerase causing inhibition until induction by the addition of IPTG. • When IPTG is added, the amount of T7 RNA polymerase increases and overcomes the inhibition by LysS.
- 14. • Protein expression from high-copy number plasmids and powerful promoters will greatly exceed that of any native host protein, using up valuable resources in the cell thus leading to slowed growth. • Additionally, some protein products may be toxic to the host when expressed, particularly those that are insoluble, act on DNA, or are enzymatically active. • For this reason, recombinant proteins are typically expressed in E. coli engineered to accomodate high protein loads using inducible promoter systems
- 16. • ompT: Strains harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the expressed recombinant proteins. • lon protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce proteolysis of the expressed proteins. • hsdSB (rB- mB-): These strains have an inactivated native restriction/methylation system. This means the strain can neither restrict nor methylate DNA. • dcm: Similarly, strains with this mutation are unable to methylate cytosine within a particular sequence.
- 17. Yeast protein expression systems – Saccharomyces cerevisiae • The highly developed genetic system, ease of use, reduced time input and costs have made S. cerevisiae an attractive organism for the expression and production of recombinant proteins. • Yeasts are able to carry specifically designed plasmids and this ability is valuable in a recombinant protein expression system. • The plasmid used consists of restriction sites that can be used to insert the gene sequence of interest. • Transformation of yeasts with the plasmid produces the desired protein and can be appropriately scaled up.
- 18. • Expression of protein using S. cerevisiae involves the following steps: • use of competent E. coli cells to take up DNA sequence of interest • integration of the DNA into bacterial genome or circularization of the DNA sequence to exist as a plasmid • selection of transformed E. coli using a selection marker (antibiotic) • expansion of selected E. coli in appropriate culture media, such as classic LB options • isolation of DNA or plasmid • transformation into yeast • screen the transformants for integration of DNA into yeast chromosome • selection and scaling-up of high expressing yeast clones in appropriate culture media • isolation and purification of intracellular/secreted proteins
- 19. • Features • low cost culture methods • suitable for both intracellular and secreted proteins • provides eukaryotic post-translational glycosylation of proteins although it results in high
- 20. • A yeast commonly used for protein production is Pichia pastoris. Examples of yeast expression vector in Pichia are the pPIC series of vectors, and these vectors use the AOX1 promoter which is inducible with methanol. • The plasmids may contain elements for insertion of foreign DNA into the yeast genome and signal sequence for the secretion of expressed protein. Proteins with disulphide bonds and glycosylation can be efficiently produced in yeast. • Another yeast used for protein production is Kluyveromyces lactis and the gene is expressed, driven by a variant of the strong lactase LAC4 promoter