recombinant proteins
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Recombinant ProteinsTRANSCRIPT
Recombinant Proteins
Amith ReddyEastern New Mexico University
Engineering Host Cells to manufacture proteins for mass production
Increasing Efficiency Transcription Systems
Activation Systems mRNA expression and stability
Translation 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
Chapter 10 Highlights
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.
FIGURE 10.15
Proteins expressed from recombinant DNA gene Reengineering of host DNA to produce desired proteins in
mass quantities
Detailed Study of Protein Expression 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.
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
Recombinant Proteins
Determining DNA, RNA, Protein sequences Sequencing techniques PCR and RT-PCR gDNA and cDNA Libraries
Cloning of correct gene into Expression Vector for enhanced production
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
Recombinant Protein Process
Fig. 10.1. Expression of Eukaryotic Gene in Bacteria - Overview
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
Prokaryotic vs Eukaryotic Cell Use in Protein Expression
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
mRNA Factors
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.
FIGURE 10.15
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
Translation Expression Vectors
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
Codon Usage Rate
13Codon Usage Affects Rate of TranslationBacteria prefer one codon for a particular amino acid to other 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.
FIGURE 10.3
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 time-dependent degradation of proteins.
Toxic Effects of Protein Overproduction
Use of vector expression system for protein production control to increase efficiency and mitigate inclusion bodies
pET Vector Expression System consists of 4 Sites: 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
Normal Function – No Protein Expresion 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
pET Vector 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
pBAD Expression System
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
Protein Stability
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
Protein Synthesis can terminate anywhere in the cell Cytoplasm, plasma membrane, extracellular matrix
Protein Secretion can be engineered to arrange for optimal destination
Use of Transmembrane proteins that are active/passive transporters
Hydrophobic signal at the N-terminal
3 Types of Secretory Systems: 1 - General Secretory System
Periplasmic Space 2 - Type 1 Secretory System
Transmembrane domain to outside of cell 3 – Type 2 Secretory System
Periplasmic Space and then outer membrane transport to outside of cell
Improving Protein Secretion
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
General 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
Type 1 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
Type 2 Secretory System
Plasmid that links or binds TWO proteins together for various purposes.
Assemblage at N-terminal or C-terminal Mainly for secretion, but also Solubility, Stability
Example: MalE Protein 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
Protein Fusion Expression Vectors
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
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Complex Fusion Expression Vector
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
Eukaryotic Cell Expression
Fig. 10.1. Post-translational Modification
Pros 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
Cons 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
Yeast Protein Expression
Fig 10.9. Protein Secretion of Yeast
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
Expression of Proteins in Insect Cells
FIGURE 10.10
Baculovirus Expression 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
Bacmid Shuttle Vector
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
Insect Cell Expression Disadvantage
Fig. 10.12
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
Geneticin – blocks protein synthesis Npt gene inactivates antibiotic
Enzymatic Selective Gene DHFR Gene knockout host cells
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Expression of Proteins in Mammalian Cells
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
Mammalian Cell Selection
FIGURE 10.13
Mammalian Shuttle Vector
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:1. Multiple vectors are used with a single gene copy of each subunit
Assembly must occur outside of cell
2. Co-expression of genes on a single vector with two separate promoters
Creates two monocistronic mRNA’s
3. 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
Expression of Proteins with Multiple Subunits in Mammalian
cells
Fig. 10.14. Expression of Multiple Polypeptides in the Same Cell
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.
FIGURE 10.15