Unind. 1 hr 2hr 3hr 4hr 1hr 2hr 3hr 4hr Unind LB Broth Miller LB Broth Lennox MW Marker Unind 1 hr 2hr 3hr 4hr 1hr 2hr 3hr 4hr 0.5 OD OD 600 Optimization of the Bacterial Expression System for LIN-12/Notch-Repeats (LNRs) from Human Notch1 and 2 Ursela Siddiqui Advisor: Dr. Didem Vardar-Ulu Wellesley College Protein expression is the translation and post-translational processing of proteins. The ability to control and manipulate this process to obtain only a desired product of a target gene is essential for biochemical research. There are four commonly used expression systems: mammalian, insect, yeast and bacteria. Due to its simplicity and high yields, biochemists heavily rely on an Escherichia coli based bacterial expression system to obtain their protein of interest for further studies. On the other hand, many parameters need to be first optimized to fully exploit this system for a particular target protein. Additionally, the protein of interest can be either soluble or insoluble under the specific expression conditions and there are advantages and disadvantages to both approaches. The first part of this project focuses on the optimization of bacterial expression for the first two tandem Lin12/Notch Repeats, LNRA and LNRB (LNRAB) from Human Notch1 and 2. Here, we report a comprehensive analysis of the optimization findings and the determined optimal set of expression conditions. The second part of the project is aimed to combine different components of the soluble and insoluble expression systems into an innovative expression and purification methodology in an attempt to alleviate some of the disadvantages each system has separately. Here, we report the preliminary results on the initial steps of this hybrid methodology and outline the remaining steps with the expected outcomes. The two major components of any bacterial expression system are: 1. A plasmid vector: a small circular molecule of double stranded DNA derived from natural plasmids that occur in bacterial cells with a piece of inserted DNA that codes for the desired protein. 2. A bacterial cell line with the desired growth and expression characteristics. Bacterial cell lines differ in the components they bring with them to the expression system. The most widely used hosts for protein expression are the BL21 strain which are deficient in both lon and ompT proteases. The cells used in the current study are all the DE3 derivatives of the BL21 line, which allows for induction by IPTG because it renders the cells under the control of the lac promoter. Further subtypes used in the current study are: 1. PlysS strains that express T7 lysozyme which stabilizes growth prior to IPTG addition. 2. Rosetta cells that have the tRNAs for AGG, AGA, AUA, CUA, CCC, and GGA. These are codons that are typically used by the mammalian systems to code for amino acids and hence E. coli normally do not contain tRNAs to translate these codons. These cell lines allow for more efficient translation of mammalian genes in the otherwise limited expression system. 3. Origami cells that have mutations which allow them to support disulfide bond formation (normally E. coli cellular environment is too reducing for this post translational modification to take place) 4. Rosetta-gami cells that have the characteristics of both the Rosetta and Origami cell lines. There are three major steps in any bacterial expression system. The first step is transformation during which the cells take up the vector. The second is growth and protein expression, where the bacteria are allowed to grow to a certain cell density and then stimulated to begin the expression of the protein. The third is isolation and purification where the desired protein is separated and collected. It is the specifics of the last two steps that differentiate one expression system from another based on the solubility of the expressed protein in the employed system. In general insoluble systems result in higher protein expression levels however there is a need to employ harsh conditions to solubilize the desired protein during purification. On the other hand, gentler purification conditions for soluble proteins make them also more prone for degradation, especially if they are small. For the first part of the project we have used several combinations of vectors and cell-lines to systematically vary several parameters within the expression system to obtain the maximum protein yield and monitored their impact on protein production via SDS-PAGE analysis. The main variables that were assayed during this study were different cell densities at the time of IPTG addition (induction), IPTG concentrations used for induction, total expression time, as well as growth media. Since each LNR is ~35 residues in length, they are extremely susceptible to degradation if produced as soluble proteins in E.coli. To thwart this problem, we decided to express a larger version of the protein initially, namely LNRAB using the pET15b vector based His-tagged soluble protein expression system and then modify it through a specific cleavage normally used during the purification steps of an insoluble expression protocol, to obtain the two individual LNRs as the desired pieces. This new hybrid strategy exploits the power of affinity chromatography for purifying the His-tagged target protein using Nickel beads. After the purification and the in vitro folding of the expressed protein, this new protocol takes advantage of a naturally occurring methionine residue between LNRA and LNRB in human Notch2 sequence to separate the polypeptide into the two individual LNRs through cleavage with cyanogen bromide. Since the individual LNRs have different physicochemical properties, after cleavage they can be separated using a reverse-phase C18 HPLC. Table 1. Optimization Parameters and Results Preliminary results from purification steps seem to indicate we were able to get enough hN2LNRAB eluted off of Nickel beads to carry out the rest of the proposed protocol. Future directions will involve evaluating the success of the new methodology by using a larger construct consisting of human Notch2 NRR. In addition, cleavage reagents such as cyanogen bromide will be used to attempt to separate constructs grown together. Even though we expressed and partially purified the target protein, our gel indicates that there are at least two significant species in the eluted fraction, so first we need to identify them and purify the desired one, then perform an in vitro folding to obtain the correct disulfide bonds and then cleave the polypeptide into two to obtain the individual LNRs. Project 1: Optimization of Bacterial Expression Multiple simultaneous small scale growths to test for various expression parameters: Overnight cultures:Day long growth and expression: 5 ml LB Broth Miller50 ml LB Broth Miller 5 µl 1000X vector/cell-line specific antibiotic 50 µl 1000X vector/cell-line specific antibiotic ampicillin kanamycin chloromphenicol Scoop of a glycerol stock or a freshly0.5 ml of an overnight culture transformed colony. Add Take hourly 1 ml sample from each flask after induction with desired concentration of IPTG Run samples on SDS-PAGE gel Figure 2: The effect of time on protein production. The band indicated by the arrow is the protein of interest seems to reach full saturation at three hours showing subtle differences in band darkness between one and two hours. Time was tested for a total of four hours. The vector and cell line used in this growth was pMML vector containing Notch1 LNR-A DNA in PlysS cells. Figure 3: The effect of IPTG concentration on protein production. The band indicated by the arrow is the protein of interest and does not get significantly darker as the concentration of IPTG increases from 0.1mM to 0.5mM. Concentrations not shown that were also tested are 0.01mM and 0.05 mM. These did not show difference either. The vector and cell line used in this growth was pMML vector containing Notch1 LNR-A DNA in PlysS cells. MW marker 0.1mM 0.2mM 0.3mM 0.4mM 0.5mM Figure 4: The effect of cell density measured at 600nm on protein production. The band indicated by the arrow is the protein of interest and is darker for the higher cell density at induction. This experiment was conducted with Origami cells containing pET32a carrying human Notch1 LNRAB DNA. Figure 5: The effect of growth media on protein production. The band indicated by the arrow is the protein of interest and does not differ in darkness from one growth media to another. The difference in growth media is LB Broth Miller has 10 g/L salt concentration whereas LB Broth Lennox has 5 g/L. The vector and cell line used in this growth was pMML vector containing Notch1 LNR-A DNA in PlysS cells. Incubate overnight at 37 °C Figure 1:Procedure for testing parameters used in bacterial expression. After adding LB Broth Miller, the appropriate antibiotics and the vector+cell lines, the solution incubates overnight at 37 C. The next day 0.5 ml of this culture is added to a multiple new flasks containing fresh LB Broth Miller and the appropriate antibiotics. Cell growth is monitored by checking samples for absorbance at 600nm and then inducing them by the addition of IPTG. Finally, hourly samples are collected after IPTG addition and run on an SDS PAGE under reducing conditions. Add supernatants to Nickel beads, incubate at 4 C for 1 hour, centrifuge and collect supernatant (FT) WASH 1: 10 mM immidazole WASH 2: 10 mM immidazole ELUTION 1: 300 mM immidazole ELUTION 2: 500 mM immidazole Add appropriate solution, centrifuge and collect supernatant Figure 6: Steps for purification after growth and protein expression. In order to lyse the cells, a sonicator is used. This solution is then centrifuged and the supernatant is collected and added to approximately 250 ml of pre- equilibrated Nickel beads. The His-tag on the protein has an affinity for these beads. To elute the protein off the Nickel beads, a buffer containing high immidazole concentration is used. Immidazole competes for the same binding site on the Nickel beads as the histidines. Notch2 LNR_AB in pET15b: Met G S S H H H H H H S S G L V P R G S H Met E N L Y F Q G A T C L S Q Y C A D K A R D G V C D E A C N S H A C Q W D G G D C S L T Met E N P W A N C S S P L P C W D Y I N N Q C D E L C N T V E C L F D N F E C Q Stop Affinity tag already on vector Used to cleave off the affinity tag from the protein construct Used to separate LNRA from LNRB 1)Induced samples were taken at time points: 1hr, 2hrs, 3hrs, and 4 hrs post induction. 2)IPTG concentrations tested ranged for 0.01, 0.05, 0.1, 0.2, 0.3,0.4, and 0.5mM. 3)Cell density as measured at 600 nm by a spectromoter ranged from 0.5 OD to 0.9 OD 4)Growth media used were LB Broth Miller and LB Broth Lennox. LB Broth Miller Contains 10g/L salt whereas LB Broth Lennox has 5 g/L Analysis of samples representing the different optimization parameters by SDS-PAGE show that regardless of cell line, much lower concentrations of IPTG than the typical values used in literature (0.5mM) and shorter induction times can be used in the expression of the tested constructs without any significant loss in production yield. We have also noted that even for 4hrs of expression, there is not a significant amount of protein degradation. Therefore, an IPTG concentration of 0.25 mM and induction time of 4 hours will be used for future large scale expressions. Furthermore, on those cell lines tested, a higher cell density does produce a more noticeable band on the SDS-PAGE. A density of 0.7 OD 600 will be used for a larger scaled growth. For the cells being discussed currently, growth media does not seem to impact protein production. LB Broth Miller will be used in any future studies as the growth media. REPRESENTATIVE DATA Project 2: Development of a new hybrid expression and purification protocol. Single large scale growth and expression under optimized conditions followed by purification. His TagTEVLNR- ALNR- B Met Recognition Site Centrifuge remainder and resuspend pellet in Lysis buffer*, sonicate and centrifuge. Repeat (SUP1, SUP2) *Lysis Buffer: 50 mM Tris H-Cl, pH mM NaCl 20 mM Imidazole, pH 8.0 NOTE: Wah and Elution buufers only differ in immadazole concentration 500 ml growth using ideal parameters found from project 1 Figure 7: His-tag purification of RosettaPlysS cells from human Notch2 LNRAB. Multiple bands indicate the purification was not complete and other components are still present. Most importantly, the multiple bands in E1 and E2 indicate this. Figure 8: Different elements of human Notch2 LNRAB construct used during the hybrid new purification protocol to obtain individual LNRs (LNR-A and LNR-B) MW Marker 1hr 2hr 3hr 4hr MW unind ind SUP1 SUP2 FT W1 W2 E1 E2 CellTime 1 IPTG concentration 2 Cell Density 3 Growth Media 1 PlysS initial gradual increase, then saturation at 3 hrs No difference when testing from 0.1 to 0.5 mM No difference OrigamiNo difference 0.7 OD showed a darker band than 0.5 OD OrigamiPlysSinitial gradual increase, then saturation at 3 hrs No difference0.7 OD showed a darker band than 0.5 OD Rosetta-gamiPlyssinitial gradual increase, then saturation at 3 hrs No difference0.7 OD showed a darker band than 0.5 OD RIPLNo difference inconclusive RosettaPlysSinitial gradual increase, then saturation at 3 hrs Subtle difference showing darker bands at 0.3 mM versus 0.1 mM 0.8 OD showed a darker band than 0.6 OD Dr. Didem Vardar-Ulu Wellesley College The Roberta Day Staley and Karl A. Staley Fund for Cancer-Related Research Vardar Ulu Lab Christina Hao Fahmi Jahufar Sharline Madera Expected MW: Expected MW: Expected MW: Expected MW: kD kD kD Expected MW: kD