Expression of cloned genes in E. coli
advantages:
– physiology, genetics, vector systems well known
– cheap & easy to grow
– generation time ~20 min.
– defined culture media
– large amounts grown in fermenters
problems with expression of foreign genes in E. coli:
1. no splicing of introns/exons
– need cDNAs for eukaryotic genes
2. foreign promoters do not work in E. coli
(unless from closely related Gram negative bacteria)
– cDNAs do not have promoters
– need promoter from E. coli gene (–35, –10 sequences)
– and bacterial ribosome binding site (Fig. 8.2)
– e.g. lac promoter – controlled by LacI repressor
– lacIq overexpresses repressor
– stops expression of toxic protein
– until induced by IPTG
– not suitable for large scale (industrial) use
– IPTG is expensive
– also controlled by catabolite repression
– glucose in media
– reduces cyclic AMP (cAMP) levels in cell
– inactivates cAMP activator protein (CAP)
– cAMP-bound form
– needed for expression from lac promoter
– expression from lac promoter
– only in media without glucose
– trp promoter – from tryptophan biosynthesis operon
– controlled by tryptophan in media
– induced under low tryptophan conditions
– stronger promoter than lac
– & has better ribosome binding site
– tac or trc promoters (1 bp difference) (Fig. 8.4)
– have –10 sequence from lac promoter
– controlled by IPTG
– have –35 sequence from trp promoter
– no catabolite repression
– or phage promoter
– e.g λ pL promoter (Fig. 8.3)
– under control of cI repressor
– use cIts857 mutant – temperature-sensitive
– induce expression at 40°C
– problem
– high
temperature activates heat shock
– proteases produced
– may degrade protein
– high temperature may denature proteins
– T7 promoter – from T7 phage
– only expressed by T7 RNA polymerase
– in vitro or in vivo
– requires cloned T7 RNA polymerase gene
– to regulate expression in vivo
– regulate T7 RNA polymerase expression
– using lac, trp, or λ pL promoter
– only cloned gene expressed at high level
– regular E. coli genes
– not recognised by phage RNA polymerase
– enzyme has high processivity
– can express longer eukaryotic genes well
3. proteases may degrade foreign proteins
– e.g. Lon protease
– degrades denatured/misfolded proteins
– including many foreign proteins
– clone in lon mutant strains
4.
proteins may form inclusion bodies
– insoluble aggregates of protein
– denatured proteins form hydrophobic interactions
advantage: – easy to purify
– lyse cells and pellet aggregates by centrifugation
problems: – must denature proteins to solubilize
– urea, detergents
– often hard to refold into active form
promoted by:
– large size of polypeptide chain
– more common if polypeptides >100 amino acids
– best expression results with smaller peptides
– e.g. insulin, human growth hormone
– large amounts of protein
– high copy number vectors, strong promoters
– high temperatures (40°C) – denature proteins
fusion proteins – may solve problems 2, 3, &/or 4 at once
– from vectors with 5' ends of E. coli genes
– multiple cloning site in gene (Fig. 8.2)
– allows in-frame cloning of cDNA
– E. coli promoter & ribosome binding site provided
– E. coli protein N-terminal domains
– may stop denaturation of protein
– e.g. lacZ fusion – pBluescript
– phoA (alkaline phosphatase) fusion
– periplasmic
protein – has signal sequence
– exported across cell membrane
(not outer membrane)
– fusions with secreted proteins exported to periplasm
– may aid in proper folding of protein
– phoA fusions with cytoplasmic genes
– often get stuck in membrane
Note – E. coli not optimal for secretion of proteins
– no type II secretion system
– most secreted proteins end up in periplasm
– protein released from periplasm using osmotic shock
– hypotonic solution – swells cytosol
– squeezes periplasmic contents out of cell
– can also add “tags” for purification of protein
– protein sequences that bind specific ligands
– purify fusion proteins
– on resin matrix with attached ligand
– e.g. glutathione-S-transferase domain (Fig.8.6)
– binds glutathione
– polyhistidine sequence – binds Ni2+
– often must remove bacterial part of fusion protein
– to get active form
– can cleave chemically
– e.g. cyanogen bromide cleaves at Met residue
– okay if no Met in active form of gene
– e.g. insulin
– can cleave with specific protease
– e.g. tobacco etch virus protease – cleaves at:
-Glu-Asn-Leu-Tyr-Phe-Gln\Gly-
possible problem if cut site not exposed
– may have to denature protein
5. codon usage
– different organisms use codons with different frequencies
– depends on GC content, methylation sites
– tRNA frequencies are optimized for codon use
– so few tRNAs
for rare codons in E. coli
– e.g. CCC, AGA, AGG
– but these are common codons in mammals
– translation of cDNA in E. coli
– will stall at these codons
– problem (especially near 5' end of gene)
– also may have problem with mitochondrial genes
– different amino acids for some codons
– can change to common E. coli codons
– site directed mutagenesis
– or synthesize artificial gene
– Genentech made insulin gene out of overlapping oligos
– based on protein amino acid sequence & E. coli codons
6.
post-translational modifications
– protease cleavage, glycosylation
– e.g. insulin
– mature protein produced by cleavage of precursor
– two chains held together by disulfide bridges
– Genentech made A & B chains as separate fusion proteins
– cloned in separate strains – expressed and purified
– cleaved fusion proteins
– mixed chains & oxidized cysteines to get mature protein
other expression systems
in vitro transcription/translation
–rabbit reticulocyte lysate
– wheat germ extract
– E. coli lysate
– low yield of protein
– okay for making small amounts of radiolabelled proteins
phage display
– expression of cloned DNA sequences as fusion proteins with M13 coat protein
– create libraries of M13 or phagemid vectors
– contain oligos cloned in 5' end of M13 coat protein gene
– get extra peptide sequences in N-terminus of protein – expressed on outside of phage particles
– used to find protein domains involved in interactions
– with other proteins or ligands
– e.g. find substrate-binding domains of enzymes
– or epitopes recognised by specific antibodies
– recognised because phage particles bind to target
– if coat protein contains proper amino acid sequence
– Dr. Scott (MBB) is expert in field
Bacillus subtilis – Gram positive bacteria
– efficient secretor of proteins
– can isolate protein from media
– B. subtilis also secretes lots of protease
– need protease mutant strains
– need Gram positive plasmid vectors (or shuttle vectors)
baculovirus vectors – derived from arthropod virus
– produce large amounts of cloned protein
– in infected insect cell lines
– media cheaper than for mammalian cell lines
problems – glycosylation differs from mammalian cells
– protein produced during lytic infection of cell line
– continuous culture not possible
mammalian cell lines
– expensive media, problems with contamination
– but always get properly folded & glycosylated proteins