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

http://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning

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