Expression of Recombinant Proteins in E.coli

• Proteins are the workhorses of the cell: structural, transport, catalysis etc • Knowledge of protein function and action can help with e.g.: drug design, engineering enzyme for use in e.g. biocatalysis

• Purification: biochemical/biophysical characterization of isolated proteins, structural studies (X-ray, NMR, cryo-EM) • In vivo studies: protein labelling techniques • Cloning and overexpression of proteins: higher yield of purified proteins, possibility to mutate amino acid residues specifically (engineering function, studying function)

• Protein-based therapeutics include: hormones, cytokines, vaccines, monoclonal antibodies • 1/3rd are produced in E. coli • Currently there are: 140 protein-based therapeutics have been approved and 500 are in clinical trials

What is it? - Purify a single protein from a mixture of proteins Why is it needed? - Separation from proteins with similar function to measure activity/function of one particular protein - Comparison of mutant proteins - Structural studies (X-ray, NMR)

• Proteins were purified from the native organism/tissue • No overexpression, most proteins typically occur at <1% of total protein: purifying Cytochrome C from ca. 1 kg Horse Heart yields 3-4 mg pure protein • With rise of recombinant DNA technology and next generation sequencing these methods are rapidly disappearing from modern research • Other developments: new overexpression systems, Tag-based protein purification, cell free expression systems • ‘old-fashioned’ methods are still used for proteins that require extensive and very specific post-translational modifications

• Developments in High throughput genome sequencing: coding sequence of our protein of interest is typically known • Gene of interest can be amplified by PCR and cloned into an protein overexpression vector: inducible overexpression results in high levels of protein in dense cultures, possibility to manipulate the gene, facilitation of the introduction of purification tags

• DNA of the protein-encoding gene (geneAB) is transcribed into mRNA • mRNA is translated into protein (ProteinAB) by the ribosome • Several regions upstream of geneAB are crucial for both transcription and translation

• 16S rRNA 3’-end complements the Shine-Dalgarno sequence, also known as ribosome binding site (RBS) • Insures that the Ribosomal complex binds in the correct location of the mRNA • Both elements need to be precisely oriented relative to the ATG start codon

• Create PCR product with restriction sites on either end • Digest PCR product and plasmid vector • Ligate digested PCR product and digested plasmid vector

• PCR (geneAB) • restriction endonuclease-based cloning • geneAB and purification tag join plasmid vector suitable for overexpression in E. coli • transformation • induce enzyme production in E.coli

• Inoculate with E. coli containing overexpression construct and grow at 37oC to mid-exponential phase (OD600 ≈ 0.5) • Induce expression of protein of interest by adding inducer followed by growth at xx oC (typically between 20-37 oC) for 4 hours to overnight

• Transcription: RNA polymerase must be able to bind to promotor and transcribe gene into mRNA • Translation: mRNA has to be translated • How is it achieved?: Insert gene of interest in between upstream regions needed for starting transcription (Promotor) and translation (Shine-Dalgarno, RBS) and terminating transcription (Terminator)

• Plasmid vectors for protein production typically contain a multiple cloning site (MCS) at the correct location relative to those 3 elements • Regulatory elements for transcription are exploited to switch protein production on/off by addition of a specific small molecule to growth medium

• Integrated into a specific protein expression plasmid • based on proteins that prevent RNA-polymerase binding to their associated promoters when inducer molecule is absent • Addition of specific small molecules to growth medium results in repression (small molecule=repressor) or induction (small molecule=inducer) of protein overexpression • If repressor can be consumed by bacterium in which the expression plasmid propagates so-called autoinduction can be used

• lac-repressor: based on the regulatory sequences and proteins of the lac-operon • PBAD promotor: based on the arabinose inducible arabad system • tet-repressor: based on the tetracycline repressor

• In absence of l-arabinose, the AraC protein is present in a form that doesn’t induce binding of RNApol to PBAD promotor site • When l-arabinose binds to AraC, it causes a conformational change ultimately facilitating binding of RNApol to promotor site • High levels of d-glucose in growth medium indirectly repress expression (via cAMP binding to CAP), even in the presence of l-arabinose • During growth E. coli consumes d-glucose, causing expression to be induced by the presence of l-arabinose (the latter is not consumed)

• Repressor-based systems regulate gene expression by effectively blocking the binding of RNA polymerase II (RNApolII) to promotor, preventing transcription • Systems consist of a repressor protein that binds to its operator sites as a dimer • operator site is located in such a way that it prevents RNApolII from binding to promotor site - no mRNA is made • Addition of inducer causes a conformational change in TetR-dimer that prevents it from binding to its operator sites • RNApolII can now bind to the promotor • RNApolII can now bind to promotor and transcribe orfAB into mRNA, producing ProteinAB

• 3 structural genes (produces a protein): lacZ – encodes for B-galactosidase, lacY - lactose permease, lacA - thiogalactoside transacetylase • 3 regulatory genes/sequences: lacP - Promoter, lacI - Repressor, lacO - Operator

• Replace lacZ-lacY-lacA cassette with GOI • Induce expression by addition of lactose or synthetic analogue Isopropyl β-d-1-thiogalactopyranoside (IPTG) • Presence of d-glucose actually enhances repression by LacI (indirectly, similar to PBAD system), even in presence of inducer • IPTG isn’t metabolised, whereas d-glucose is, so autoinduction can be used for lac-based systems as well

• Inoculate medium with consumable repressor (d-glucose) and inducer (e.g. IPTG) with E. coli containing overexpression construct and grow at xx oC (typically between 20-37 oC) • During growth, repressor (d-glucose) is consumed whereas level of inducer (e.g. IPTG) stays the same • Ultimately all repressor (d-glucose) is consumed (starting levels are chosen in order to time this during mid-exponential phase) and overexpression will start without ‘automatically’ (normally inducer is added manually)

• use a ‘two-step’ expression system • Gene cloned in plasmid MCS (insert) is transcribed by a recombinant T7 RNA polymerase • Expression of T7 polymerase from chromosomal location that is under LacI control • Protein is only expressed in E. coli strains that express T7 RNA polymerase • Most commonly used: E. coli BL21 (DE3) • All ‘genetics’ are carried out in different E. coli strains more suited for DNA purification

• LacI repressor protein blocks transcription at 2 points: lacO site on host genome thus repressing transcription of the T7 RNA polymerase gene, lacO site on the pET-vector thus repressing transcription of the recombinant gene • However, even in absence of inducer (IPTG) there can be some expression of T7 RNApol from the lac promotor on E. coli BL21(DE3) genome • Expression of T7 lysozyme from pLysS plasmid actively blocks T7 RNApol, fixing the ‘leakiness’ of the system • IPTG induction produces excess T7 RNApol, overcoming block from limited amount of T7 lysozyme

• Must be 18 nt in length • Must finish at the 3’ end in either G or C • 𝑇𝑚 (midpoint melting temperature in oC) values of the primer pair must be within 5oC of each other • annealing temperature for PCR is 5oC less than the lowest 𝑇𝑚-value of the primer pair

• 𝑇𝑚=69.3+0.41×𝐺𝐶%−650/(𝑝𝑟𝑖𝑚𝑒𝑟 𝑙𝑒𝑛𝑔𝑡ℎ (𝑛𝑡))