Protein Expression and Purification

Protein Expression and Purification Overview Whether you are starting off with a recombinant protein which is produced in E. coli, or trying to isolate a protein from some mammalian tissue, you are typically starting with gram quantities of a complex mixture of protein, nucleic acids, polysaccharide, etc. from which you may have to extract milligram quantiles of desired protein at high purity, and hopefully with high yield Protein purification can be thought of as a series of fractionation steps designed so that at the end: ‣ The protein of interest is found almost exclusively in one fraction (and with good yield) ‣ A significant amount of the contaminants can be found in a different fraction Importance of the right cell line The phoA gene in the pET20 plasmid is transcribed by T7 RNA polymerase. This is an RNA polymerase from a bacteriophage not the endogenous E. coli RNA polymerase BL21 (DE3) cells have the gene to make T7 RNA polymerase inserted into their chromosome. The T7 RNA polymerase gene is under the control of a modified lac opera and its expression is "Off" until we remove the transcription controls which is also called inducing transcription or gene expression Researchers took advantage of the native lac operon which regulates sugar metabolism in E. coli and inserted this operon in front of the T7 RNA polymerase gene to regulate its expression Induction of transcription (gene expression) Under typical growth conditions with a large availability of glucose, a lac repressor protein binds to the endogenous lac operator which is upstream of genes necessary for metabolism of lactose. The cell does not make these proteins until it needs to metabolize lactose In the BL21 (DE3) cells we are using in lab, the T7 RNA polymerase that was inserted into the E. coli chromosome is also under control of a modified lac operon This means the repressor protein also binds to the modified lac operator upstream of the T7 RNA polymerase gene. This physically blocks transcription of the T7 RNA polymerase gene as the E. coli’s endogenous RNA polymerase can not bind well to the promoter to start transcription In E. coli, when cellular and environmental glucose levels are low and lactose is available, the E. coli will switch to using lactose as the primary sugar source for metabolism Allolactose binds to the repressor protein on the lac operon causing a conformation change in the repressor which results in its release from the operator allowing E. coli RNA polymerase to access the genes downstream of the lac operator In the lab, to remove the repressor protein and allow transcription of the T7 RNA polymerase gene, the media is typically supplemented with a chemical which mimics allolactose such as IPTG After the T7 RNA polymerase mRNA is translated, the active polymerase can begin transcribing the phoA gene on pET20 Auto-induction media An alternative induction method is to use what is called “autoinduction media". Autoinduction media contains a specifically proportioned mixture of glucose and lactose. Glucose is the preferred sugar for E. coli and the cells Will metabolize glucose first Then, as glucose runs out, the cells begin to take up lactose to use in metabolism. This starts the endogenous process of converting lactose to allolactose to activate the lac open by removing the repressor protein Some of the allolactose will also bind to the repressor protein that is bound to the operator upstream of the T7 RN polymerase gene. This removes the transcriptional block and the cells begin to express and make T7 RNA polymerase which can now bind to our pET20+phoA plasmid and express alkaline phosphatase Cell lysis and protein extraction E. coli has an inner and outer membrane. The space between the membranes is called the periplasmic space. It is a gel-like matrix and it is where the peptidoglycan layer is found Bacterial alkaline phosphatase contains a signal peptide encoded by an N - terminal signal sequence. This 21 amino acid peptide initiates translocation of alkaline phosphatase from the cytoplasm to the periplasmic space The pET20 plasmid contains a second signal sequence called pelB signal sequence. This enhances the translocation of our recombinant alkaline phosphatase to the periplasm Having Our protein in the periplasm is beneficial for a few reasons: (1) movement of our recombinant protein to the periplasm will result in removal of the N-terminal methionine when the signal sequence is cut. This is similar to endogenous alkaline phosphatase (2) periplasm is conducive to disulfide formation. Alkaline phosphatase has 4 disulfide bonds (3) periplasm has less proteases than the cytoplasm (4) easier to extract proteins from the periplasm than the cytoplasm Detergents are amphipathic molecules, meaning they contain both a non-polar "tail" having alipathic or aromatic character and a polar "head" Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic. Sodium dodecyl sulfate (SDS) is an anionic, denaturing detergent Moderate concentrations of mild detergents compromise the integrity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form Using certain buffer conditions, various detergents effectively penetrate between the membrane belayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins Immobilized metal-affinity chromatography (IMAC) Affinity chromatography requires that you know something specific about your protein -- that it has a specific tag engineered into the sequence, you know the ligand it binds or that you have a specific monoclonal antibody that interacts with your protein Immobilized metal-affinity chromatography (IMAC) is a widely used method to purify recombinant proteins containing a short affinity tag consisting of polyhistidine residues If the phoA gene was inserted correctly into the plasmid, once the plasmid was transcribed and translated the resulting alkaline phosphatase protein should have what is called a six-histidine tag on its C - terminus Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal The Importance of Histidine The importance of histidine Histidines importance is due to its structural and chemical properties. The side chain of histidine is an imidazole group and has a pKa of around 6 which is close to physiological pH, so small changes in the environmental pH can readily change the histidine protonated state Histidine in metal coordination Metal coordination bonds are a unique class of chemical interactions that make up a grey area of the normally distinct line between covalent and non-covalent bonding The coordination bond between the metal and the nitrogen of histidine involves sharing of electrons between the p orbital containing the lone pair on the ligand (histidine) and d orbitals on the metal