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Recombinant Proteins
- 1. Recombinant Proteins Amith Reddy Eastern New Mexico University
- 2. Chapter 10 Highlights Engineering Host Cells to manufacture proteins for mass production Increasing Efficiency Transcription Systems Translation Activation Systems mRNA expression and stability Translational Control Systems Codon Optimization Protein Stability and Purification Comparisons of Different Host Cell Expression Systems Pre- and Post-Translational Modification Systems Multiple Expression Systems
- 3. FIGURE 10.15 Comparison of Recombinant Protein Expression SystemsEach protein expression system falls on a continuum of worst to best for characteristics such as speed, cost, glycosylation, folding, and government regulations. Transgenic animals (rabbit) and transgenic plants (plants) are discussed in Chapters 14 and 15The other symbols include mammalian cultured cells, insect cell culture, yeast, and bacteria.
- 4. Recombinant Proteins Proteins expressed from recombinant DNA gene Detailed Study of Protein Expression Reengineering of host DNA to produce desired proteins in mass quantities DNA Techniques RNA Techniques Protein Expression Techniques Protein Purification and Production Large Scale Protein Production Clinically Relevant Proteins Insulin, Interferon s, IL-2, Somatotropin, Erythropoietin, etc.
- 5. Recombinant Proteins Pros Pathway engineering is very specific for easy manipulation depending on host cell and protein desired. Greater copy number of genes results in higher quantity of product Can use high-copy plasmids Prevent plasmid loss by genome integration of DNA Cons Large scale production and purification is extremely difficult and precise High-copy plasmids may be unstable or redundancy may occur Can be difficult to integrate multiple copies of gene into host genome due to unreliability of multiple gene copy integration
- 6. Recombinant Protein Process Determining DNA, RNA, Protein sequences Cloning of correct gene into Expression Vector for enhanced production Sequencing techniques PCR and RT-PCR gDNA and cDNA Libraries Restriction Endonuclease Digestions Gene Intregation and Ligation into Vector Transformation of Vector into Host Cell and Expression Cold Shock and Ca Treament for Transformation Gene Intregation into gDNA Heat Shock
- 7. Fig. 10.1. Expression of Eukaryotic Gene in Bacteria - Overview
- 8. Prokaryotic vs Eukaryotic Cell Use in Protein Expression Prokaryotic Cells Easiest cells to grow and genetically manipulate Antibiotic resistance genes for increased selectivity of transformed bacteria Lack before and after-translation protein modification pathways for correct protein manufacturing Eukaryotic Cells Not all genes are able to be expressed in prokaryotic cells Has all necessary promoters and terminators in gDNA already
- 9. mRNA Factors Strength between mRNA Ribosome Binding Site and Ribosome interaction mRNA Stability and Structure Codon usage Prevention of mRNA secondary structure overlap or folding Correct formation of poly A tail and methyl-G cap
- 10. FIGURE 10.15 Comparison of Recombinant Protein Expression SystemsEach protein expression system falls on a continuum of worst to best for characteristics such as speed, cost, glycosylation, folding, and government regulations. Transgenic animals (rabbit) and transgenic plants (plants) are discussed in Chapters 14 and 15The other symbols include mammalian cultured cells, insect cell culture, yeast, and bacteria.
- 11. Translation Expression Vectors Vector provides the most optimal ribosomal binding site Strong consensus RBS and 8 bp space between RBS and Start codon for increased binding affinity and improved translation mRNA may back onto RBS region depending on sequence
- 12. Codon Usage Rate Engineering of DNA sequence for codon optimization Alter DNA sequence for improved translation effeciency Can limit translation if tRNA anticodons used for amino acids are not in abundance Ex. Lysine encoded by AAA 25% and AAG 75%. Figure 10.3 has E. coli waiting on UUU tRNA since it mainly uses AAG as primary codon. Directly supply rare tRNA for increased translation Can be very expensive depending on scale of production
- 13. FIGURE 10.3 Codon Usage Affects Rate of TranslationBacteria prefer one codon for a particular amino acid to other 13 redundant codons. In this example, the ribosome is stalled because it is waiting for lysine tRNA with a UUU anticodon. Escherichia coli does not use this codon very often and there is a limited supply of this tRNA.
- 14. Toxic Effects of Protein Overproduction Overproduction of proteins may condense into an aggregate of misfolded and nonfunctional proteins called Inclusion Bodies Inclusion bodies result in a decrease in efficiency and waste of resources Results from limitation in protein processing and natural timedependent degradation of proteins.
- 15. pET Vector Expression System Use of vector expression system for protein production control to increase efficiency and mitigate inclusion bodies pET Vector Expression System consists of 4 Sites: Normal Function – No Protein Expresion Site of transcription with lac operon and gene of interest Origin of Replication and Antibiotic Resistance Gene Lac I for production of Lac operon repressor protein Lac I protein represses transcription by preventing T7 RNA Polymerase expression Altered Function – Protein Expression IPTG is added to induce protein expression IPTG binds to Lac repressor protein and expresses T7 RNA Polymerase for transcription
- 17. pBAD Expression System Expression system based on Arabinose Operon Normal Function – OFF AraC regulatory proteins bind O2 and O1 sites and create dimer Addition of Arabinose – ON AraC binds to I site and activates transcription Transcription increase is dose-dependent
- 18. Protein Stability Factors in Protein Stability and Degradation Natural Degradation or time left unprocessed Overall 3D Structure N-end Rule Prokaryotes – Val, Met, Ala, etc – 20 hr, and Arg – 2 min Humans – Val – 100 hr, Met/Gly – 30 hr, and Glu, Arg – 1 hr Easy to alter through DNA Sequence to produce longer lasting free proteins Pest Sequences Regions rich in (P) Proline, (E) Glutamine, (S) Serine, and (T) Threonine Very recognizable by proteosomes Most difficult to alter these sequences due to internal sequence change that can disrupt final protein function or disrupting protein synthes Alter final protein function or make protein nonfunctional Disruption of protein synthesis or make protein unstable during synthesis
- 19. Protein Stability Addition of Moleculer Chaperones to mitigate formation of inclusion bodies Molecular chaperones bind free amino acids of the growing polypeptide chain before folding
- 20. Improving Protein Secretion Protein Synthesis can terminate anywhere in the cell Protein Secretion can be engineered to arrange for optimal destination Periplasmic Space 2 - Type 1 Secretory System Use of Transmembrane proteins that are active/passive transporters Hydrophobic signal at the N-terminal 3 Types of Secretory Systems: 1 - General Secretory System Cytoplasm, plasma membrane, extracellular matrix Transmembrane domain to outside of cell 3 – Type 2 Secretory System Periplasmic Space and then outer membrane transport to outside of cell
- 21. General Secretory System Transports protein into periplasmic space Allows protein extraction harvest from cell Aggregate of Inclusion bodies may occur if there is overproduction of protein Increase of secretory proteins into inner membrane can be used to decrease inclusion bodies
- 22. Type 1 Secretory System Transport of protein through periplasmic space to the outside of the cell by a transmembrane protein that spans entire membrane Protein may have hydrophobic signal sequence at N-terminal for simple transport Protein Fusion may be used to transport across Fusion of Normal protein and Bacterial protein that can be transported across membrane Binding maltose protein to normal protein for transmembrane delivery Cleave maltose after transport by proteases
- 23. Type 2 Secretory System Two Step System Transport of protein into periplasmic space by general secretory system Transport of protein from periplasmic space to the outside of cell by an outermembrane protein Combination of general and Type 1 systems Specific export of protein outside of cell
- 25. Protein Fusion Expression Vectors Plasmid that links or binds TWO proteins together for various purposes. Example: MalE Protein Assemblage at N-terminal or C-terminal Mainly for secretion, but also Solubility, Stability Protein fused to MalE within cell Transport of fusion protein to Periplasmic space by maltose induction Pre-made Fusion Expression Vector Mix and Match Fusion Proteins through pBAD expression control
- 26. Protein Fusion Expression Vector Examples Simple Protein Fusion Vector Single Vector with attachment to thioredoxin protein CM4 is GoI ProAsp gene is for peptide cleavage site His-tag is for purification http://www.springerimages.com/Images/LifeSciences/1-10.1007_s10529-007-9351-4-0
- 27. Complex Fusion Expression Vector http://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning
- 28. Eukaryotic Cell Expression Post-translational modification systems in Eukaryotes Novel Amino Acids in protein sequence Glycosylation for cell surface recognition and function retention Addition of other chemical groups Fatty Acid Chains – lipids Acetyl Groups Phosphate Groups – DNA , RNA, phosphorylation Disulfide Bonds Cleavage sites
- 29. Fig. 10.1. Post-translational Modification
- 30. Yeast Protein Expression Pros Cons Similar to bacterial protein expression Naturally occuring plasmid Secretes few proteins for easy purification of recombinant protein Able to carry out many post-translational modifications Loss of expression plasmids in large bioreactors Only glycosylates secreted proteins (can be altered) Addition of signal sequence to recombinant protein for secretion and purification (Fig 10.9) Similar to protein fusion
- 31. Fig 10.9. Protein Secretion of Yeast
- 32. Expression of Proteins in Insect Cells Insect cells are simple and cheap to grow with many of the added benefits of using mammalian cells Vectors are Baculoviruses Baculovirus infects insect cells and take control of cell for viral protein production After host death, baculovirus embeds viral particles in protein matrix (capsule) called Polyhedrons Polyhedrin is not needed. Transfer gene of interest to Baculovirus at this site Main baculovirus is the Multiple Nuclear Polyhedrosis Virus (MNPV) Broad spectrum baculovirus High yield of polyhedrins
- 33. Baculovirus Expression Vector FIGURE 10.10
- 34. Bacmid Shuttle Vector Baculovirus expression vectors may give undesirable results Bacmids created as a shuttle vector for alternate use of infecting insect cells Baculovirus-plasmid hybrid Contains E. Coli origin, cloning site, and antibiotic resistance site Allow bacmids to survive in E. coli and infect insect cells Figure 10.11
- 35. Insect Cell Expression Disadvantage Glycosylation pathway is different in Insect Cell lines in mamalian cell lines Insect Cells – Mannose derivative pathway Mammalian Cells – Full glycosylation pathway of sialic acid derivatives Fig. 10.12
- 36. Expression of Proteins in Mammalian Cells Most complex method of engineering for mammalian cells Mammalian Shuttle Vectors include: Bacterial origin of replication and antibiotic resistance Selection at prokaryotic level Strong viral or mammalian promoters Multiple cloning sites Types of selective genes for mammalian cell growth Antibiotic Selective Gene Enzymatic Selective Gene Geneticin – blocks protein synthesis Npt gene inactivates antibiotic k DHFR Gene knockout host cells
- 37. Mammalian Cell Selection Three Types of selective genes for mammalian cell growth Antibiotic Selective Gene Geneticin – blocks protein synthesis Npt gene inactivates antibiotic Enzymatic Selective Gene Host Cell knockout of DHFR gene DHFR – cofactor of folic acid and inhibited by methotrexate DHFR gene is included on plasmid Methotrexate inhibition for high-level expression selection Metabolic Selective Gene Glutamine synthetase enzyme is included in shuttle vector Select cell lines by addition of methionine sulvoximine Mammalian cell selection with multicopy plasmids
- 38. Mammalian Shuttle Vector FIGURE 10.13
- 39. Expression of Proteins with Multiple Subunits in Mammalian cells Expression of proteins with multiple subunits can be difficult to produce because assembly of protein outside of cell is very difficult Three Methods for multiple subunit expression: Multiple vectors are used with a single gene copy of each subunit 1. 2. Co-expression of genes on a single vector with two separate promoters 3. Assembly must occur outside of cell Creates two monocistronic mRNA’s Co-expression of genes on a single vector with a single promoter and an IRES between genes Creates one polycistronic mRNA Two ribosomes read the same mRNA for multiple subunit translation
- 40. Fig. 10.14. Expression of Multiple Polypeptides in the Same Cell
- 41. FIGURE 10.15 Comparison of Recombinant Protein Expression SystemsEach protein expression system falls on a continuum of worst to best for characteristics such as speed, cost, glycosylation, folding, and government regulations. Transgenic animals (rabbit) and transgenic plants (plants) are discussed in Chapters 14 and 15The other symbols include mammalian cultured cells, insect cell culture, yeast, and bacteria.