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MI324 LAB RECOMBINANT PROTEIN PRODUCTION (RPP)  Recombinant proteins are proteins expressed in a non-natural host, e.g., human insulin, expressed from a cloned human DNA sequence in Escherichia coli.  RPP is widely used in drug delivery, biopharmaceuticals, engineering, food industry, etc.  We use various RPP hosts, mostly E. coli, to produce proteins for use in: - Functionalising cardiovascular stents with antibody fragments to work better in vivo - Producing nature-derived surgical glues - Drug delivery: e.g., to lungs, spinal cord - Disease detection/ diagnosis  The most commonly used recombinant hosts are bacteria (typically E. coli), fungi and yeasts (e.g., Saccharomyces cerevisiae) and mammalian cells (e.g., Chinese hamster ovary).  In all RPP systems, some optimisation is typically required to identify the ideal conditions for the host cells to express, fold and possibly secrete the protein of interest.  Optimisation of protein production can involve manipulation of - Temperature, growth medium - The host strain – genetically - Duration of production - The protein itself In this lab series, we investigate the optimal length of induction time for a recombinant antibody fragment expressed in E. coli by analysing cell samples using SDS-PAGE and staining. We will also study how to pour SDS-PAGE gels and carry out immunoblot detection of proteins.

SDS: (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) – A discontinuous electrophoretic system commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. Reliable method for determining the molecular weight (MW) of an unknown protein, since the migration rate of a protein coated with SDS is inversely proportional to the logarithm of its MW. 1. The method initially denatures the proteins that will undergo electrophoresis. Protein of interest in this lab series: anti-SARS-CoV-2 scFv scFv: single-chain variable fragment. scFVs are a type of recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody.  SARS-CoV-2 is the causative agent of COVID 19.  We have expressed virus proteins; isolated antibody fragments – scFvs – against them using phage display technology; and are incorporating them into highly sensitive, handheld diagnostic instrument for rapid, point-of-care testing.  We need to optimise production of the scFvs in E. coli so that we can purify them in sufficient amounts to develop (and scale up) the diagnostic device.  Common problem in RPP: when E. coli express recombinant proteins, they can undergo stress responses and, ultimately, cell lysis due to the strain on their normal protein production and folding machineries.  In this work, expressing cells were harvested by centrifugation after 0, 3, and 16 hours of protein expression.  Goal is to identify the optimal expression period for E. coli cells producing the scFv (and confirm the predicted mol. Weight of the scFv).

BACKGROUND 1. PAGE: Polyacrylamide gel electrophoreses  Polyacrylamide is a chemically inert polymer that can be made with different pore sizes to separate proteins. It is formed by monomers of acrylamide, which polymerises into long strands, and bis-acrylamide, which cross-links the strands to form a porous gel matrix.  Polyacrylamide gels are typically poured in two parts: at the top is a stacking gel, with larger pore sizes, and below a resolving gel, where migration proportional to protein mass happens. (The purpose of stacking gel is to line up all the protein samples loaded on the gel, so that they can enter the resolving gel at the same time. The resolving gel is to separate the proteins based on their molecular weight).  When samples are loaded into a polyacrylamide gel, they typically fill the well - up to 1cm high. To avoid having broad, smeared bands moving down the gel, the stacking gel functions to “focus” the protein samples in very narrow bands before they enter the resolving gel.  This is achieved by a discontinuous buffer system, in which the pH differs between the stacking gel, resolving gel, and buffer in contact with the electrodes. 2. SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis. Negatively charged detergent SDS Used to denature and linearise the proteins Confers a uniform negative charge of proteins.  This is a technique that is used to analyse the purity of proteins and to estimate their apparent molecular weight – similar manner to agarose gel electrophoreses in the analysis of DNA or RNA.

 SDS is an ionic detergent that binds proteins and breaks the non-covalent bonds in the protein structure that determine its conformation (folding). Proteins therefore become linearised in SDS solutions.  As amino acids in the protein are linked covalently, by peptide bonds, the protein doesn’t disintegrate into individual amino acid residues. Instead its α helices, β sheets, etc become denatured and the protein forms a linear “polypeptide”.

Two important principles of SDS in terms of its binding to proteins: 1. As it is negatively charged, the resultant “micellar chains” (linearised protein chains with attached SDS molecules) mask any original charges on amino acid and migrate towards the positive electrode if placed in an electric field. 2. As it binds proteins uniformly, the degree of migration of most proteins is proportional to their molecular weight. So proteins placed in an electric field can be ‘”sized” and their migration compared to that of molecules of known sizes. In SDS-PAGE, the molecular weight standards are purified proteins of known molecular weights. These can vary in size range, (pre)staining, etc – lots of commercially available variations.

DESCRIPTION 1. Sample preparation for electrophoresis

Essentially destruction of the tertiary structure of the protein and masking of its native charge so that the protein molecules will be separated based on their size. So, you add sample buffer containing: 1. SDS will denature proteins, allowing them to be separated based on their mol. Weight. 2. A reducing agent (dithiothreitol [DTT] or β-mercaptoethanol) to break disulphide bonds. 3. A dye (bromophenol blue), to track migration of the samples in gel and allow you to load samples and track electrophoresis. 4. Glycerol (for ease of loading samples) 5. Heat from 60-100 °C – helps the process of protein denaturation. Keep samples on ice prior to loading. Step 1: Pouring the SDS-PA gel  Assemble the gel plates as described on the instruction video. Insert into the casting frame and place in the casting stand.  When the sandwich is ready on the casting try, prepare a sealant gel mixture, ensuring TEMED (pH 8.8) (tetramethyl ethylenediamine) is added last as this initiates the polymerisation reaction.  Immediately upon adding TEMED, pipette 1ml of the sealant gel slowly down the side of the gel space and allow to run across the width of the gel plates.  After the sealant gel polymerises (10-20 mins), insert the comb between the glass plates and make a mark on the front plate approx. 1cm below the bottom of the teeth. Remove the comb.  Prepare the resolving gel and add TEMED (pH 8.8) last. Mix solution well.  After checking that the sealant gel has polymerised, slowly pipette the gel solution into the glass sandwich using a 1ml pipette until it reaches the mark on the plate.  Pipette water very slowly down the side of the gel sandwich until it covers the gel surface. Rock the sandwich gently to

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eliminate bubbles and ensure a smooth interface between the resolving and stacking gels. While the resolving gel is polymerising (~15-30 min), prepare the stacking gel solution. Note the different Tris buffer to be used in the stack solution. (1.0 M Tris pH 6.8) Do not add TEMED until the resolving gel has polymerised. When the resolving gel has polymerised, pour out the overlaid water and add TEMED to the stacking gel mixture. Pipette the gel solution into the glass sandwich until it reaches the top of the plates. Carefully insert the comb, ensuring not to capture air bubbles under the teeth. Allow the gel to polymerise, which should take ~15-30 min.

Step 2: Storing the gel for electrophoresis  Disassemble the gel casting unit and do not remove the comb. Measure and record the dimensions of the resolving gel.  Wrap the gel in tissue paper, concentrating on the comb end.  Gently saturate the tissue paper with water. Wrap in cling film or seal in a sandwich bag to retain moisture. Gels will be stored at 4°C for use in the next lab session. PROTEIN STAINING Most proteins have no colour so after electrophoresis, proteins in the gel must be stained for visualisation. Most commonly used stains are Coomassie blue and silver stain. Practical 1: Running SDS-PAGE gels; protein staining Step 1: Preparation of bacterial samples for electrophoresis  E. coli cell solutions harvested 0, 3 and 16 h after induction of protein expression are provided. Each pellet was generated by centrifugation to pellet the bacterial culture, discarding the

supernatant, and resuspending the cells in 1/10 volume of PBS buffer.  To the E. coli cell samples and the BSA control, add 30 µl of reducing buffer (gel loading buffer) and vortex well. The molecular weight standards are pre-prepared with gel loading buffer by the supplier so no buffer needs to be added. (Loading buffer increases the density of the sample; denser objects sink, so adding loading buffer to the DNA samples will enable the DNA molecules to sink into the wells in the gel in preparation for gel electrophoresis, and they provide dye markers that allow you to see the sample as you load it and provide you with information regarding the separation of samples on the gel as it is running.  Incubate tubes for 5 min at 95°C (in water bath or on heating block), followed by 1 min on ice.  Vortex tubes briefly. Spin down briefly and store on ice prior to loading. Step 2: Gel electrophoresis  For this year’s labs, you will electrophorese samples in pre-cast commercial gels.  Remove the gel from its pouch by tearing at the notch. Note the lanes are outlined for ease of sample loading.  Remove the green tape from the bottom of the gel.  Assemble the centrepiece of the gel running system, ensuring the shorter plate is facing into the buffer.  Insert the gel sandwich into the running chamber.  Fill the chamber between the gel sandwiches with the 1x running buffer provided.  Slowly and gently, remove the comb, taking care not to rupture the walls of the wells.  Wash out the wells with buffer using a Pasteur pipette before loading samples.

 Load 20 µl of each sample per well. Note any problems in loading of samples (e.g. cross-over of samples between lanes during loading).  Lane 1- protein marker, L2- control, L3- T0, L4- T3, L5- T16, L6empty, L7- protein marker, L8- T0, L9- T3, L10- T16. Molecular weight markers, or ladders, are a set of standards that are used for determining the approximate size of a protein run on an electrophoresis gel. These standards contain pre-determined protein sizes and concentrations.  Add running buffer to the gel tank until it covers the bottom of the gels (and the electrodes).  Run the gel at 250 V for 30 min. Step 3: Preparation for Western blotting  Weigh out and prepare 25 ml of 5% skim milk powder in TBS (tris-buffered saline; provided). Step 4: Coomassie staining  When the electrophoresis has finished, switch off power and remove the electrodes. Disassemble and remove the gel sandwich from the electrophoresis tank.  Before opening the plates, measure the gel half – resolving gel only – to be immunoblotted (the one not containing the control). Cut six rectangles of the filter paper provided and one of nitrocellulose membrane to the same dimensions.  Ease apart the plates at the four arrows marked on the plates, using the tool provided. Remove the stacking gel as this makes blotting easier.  Using the same tool, cut the gel vertically along the empty sample lane, leaving one gel half to be immunoblotted (one without the control) and the other to be stained with Coomassie blue (containing the control).  Transfer the half of the gel to be stained into the sandwich box provided and rinse twice using 15 ml water, taking care not to discard the gel when you pour away the water

 Carefully add 5 ml of the Coomassie blue staining solution provided. Rock at room temperature for 5-10 min.  Observe the banding pattern that appears in each of the samples. Stop the staining by pouring away the stain and rinsing – and storing – the gel in water. In Lane 1, the protein marker bands will allow estimation of the size of protein bands in the other lanes. In Lane 2, there should be a single band in the BSA control lane as it contains a single protein. The contents of lanes 3,4,5 represent the proteins in cells at time 0, time 3 hours and time 16 hours. Ideally you should see a thick band corresponding to the size of the protein that was expressed in at least one of the times 3 hours and time 16 hours lanes. In order to confirm this is the protein of interest, immunoblotting will be carried out on the same samples on the other half of the gel. Step 5: Western blotting  Using the other half of the gel, assemble the blotting sandwich on a clean, flat surface, as seen in the video: three sheets of filter paper, pre-wet in transfer buffer, followed by nitrocellulose membrane, also pre-wet. Roll out any air bubbles using a clean test tube to ensure a homogenous transfer.  Wet the gel on the plate using water. Ease the gel off the plate and place it on the membrane, being careful to match up the edges of gel and membrane precisely.  Complete the immunoblot sandwich with the three remaining sheets of filter paper in turn, each pre-wet in transfer buffer. Roll out any air bubbles gently.  Transfer the blotting sandwich to the immunoblotting chamber.  Proteins will be transferred to the membrane at 120 mA over 45 min, after which the blotting sandwich will be disassembled. The membrane will be incubated in your blocking solution for 1 hour at room temperature with gentle rocking, washed with 1x TBS buffer, and stored at 4°C until the next practical session.

Practical 2: Western blotting; immunodetection of recombinant proteins Immunoblotting With SDS-PAGE and protein staining, ALL proteins are stained. This can make it impossible to detect a specific protein of interest in a complex, heterogeneous mixture, e.g. a cell lysate or a lowabundance cytokine. Immunoblotting is the use of antibodies to detect specific proteins in complex mixtures. Proteins are transferred onto a membrane (PVDF, nitrocellulose) – this can be by capillary action but is usually carried out by electroblotting – and probed with labelled antibodies that bind specifically to the target. After transfer of the protein mixture onto the surface of the membrane, proteins are accessible to antibodies, which bind and report their presence – similar to an ELISA. The final result can be reported in the form of colour development, or a radioactive, fluorescent or (chemi)luminescent signal. The main advantages of immunoblot detection of proteins over simple staining are: 1. Specificity – can find your protein in a complex mixture 2. Sensitivity – amplification of signal built into the detection / reporting method Why is this important? Using stains, ALL proteins are stained - so you can’t identify yours: (or even tell if it has been produced) While most proteins have no colour, our mol wt markers are stained: And as we denatured everything, we can compare to estimate molecular weights. But also, blotting amplifies the signal for increased sensitivity of detection Step 1: Blocking the membrane

 After the 45-min electro-transfer was complete, the transfer sandwich was disassembled for you and the nitrocellulose membrane removed.  The membrane was added to a clean sandwich box containing the prepared blocking solution and incubated for 1 h at room temperature with gentle rocking.  After rinsing in TBS, membranes were stored at 4°C for analysis. Step 2: Immunodetection  Wash the membrane once in 15 ml of 1x TBS buffer by adding, swirling and discarding the buffer solution.  Add 15 ml of the 1:5,000 dilution of anti-hexahistidine horse radish peroxidaseconjugated murine antibody provided (previously diluted in 1x TBS containing 1.5% bovine serum albumin – BSA) to the membrane. Rock at room temperature for 1 hour.  Pour the antibody solution back into its original container – DO NOT DISCARD! After two washes of the membrane in 1x TBS for 2 min each, pipette 750 µl of the TMB substrate provided over the surface of the membrane. Continue to pipette the substrate over the membrane and to rock the membrane in the substrate solution for 1-5 min until protein bands become clearly visible.  Stop colour development by rinsing the membrane in distilled water.  All stained gels and membranes will be photographed and pictures provided via Blackboard. Ensure you have written your group’s name and order of loading on the board. Loading order of all gels will also be provided on Blackboard so you can interpret all results from around the laboratory. Because the antibody is specific for the protein of interest, the size and intensity of the brownish bands indicates the quantity of the protein produced.

At time 0 hours, no protein is expected as expression has not begun. Looking at time 3 hours and time 16 hours samples you should be able to determine the optimal length of induction time for this expression culture....