Protein Isolation Using Peptoid Based Affinity Chromatography

Protein purification is essential for advancements in biotechnology. There are several different methods employed in purifying a particular protein from a complex sample such as a cell lysate. These methods take advantage of differences in the size, charge or binding affinity of the protein. One such method is affinity chromatography which utilizes the binding affinity of a protein toward a certain ligand to purify a protein. This is usually used as a final step to extract the desired protein after the mixture has undergone other purification steps to remove unwanted materials. The goal of this project was to develop a one-step peptoidbased protein purification method. Poly-N-substituted glycines, or peptoids, were developed in the early 1990s and have been shown to have many biological applications. Peptoid side chains can be manipulated for unique circumstances by utilizing any free amine in synthesis. This is an advantageous quality in the determination of protein ligands. This study investigated using peptoids as a specific and efficient one-step process for purification methods. It showed there is potential of proteins binding to peptoids by determining protein concentration changes caused by incubation studies. However, these results could not be verified. Introduction and Background One type of protein purification technique is column chromatography. It works by having a stationary phase that is a solid adsorbent, and a mobile phase that is a liquid. The liquid is sent through a column and the desired component is absorbed by the solid. This efficient method is generally used to separate all proteins from cell lysate. One method of the stationary phase utilizes a technique known as immobilized metal ion affinity chromatography (IMAC). IMAC is a process that uses proteins that have an affinity towards metal, usually nickel, copper, cobalt or zinc. These proteins then bind to the solid adsorbent that is composed of microbeads coated in the corresponding metal. Some proteins are not attracted to metal and must be altered. This is done by using recombinant DNA techniques to add a tag that has an affinity towards metal to a particular protein. A disadvantage of using this technique is that IMAC alone cannot provide both efficiency and specificity. Efficiency refers to its ability to extract all of the desired protein, and specificity meaning its capability to extract only the desired protein. It has been suggested that a primary purification step known as aqueous two-phase extraction (ATPE) be used. ATPE is used as a primary step because it is efficient meaning it captures all of the proteins. IMAC must then be used to specifically purify the sample in order to get the desired component.3 Poly-N-substituted glycines or peptoids were developed in the early 1990’s and have been shown to have many biological applications. Peptoids have a structure similar to that of a peptide (see figure 1); however, peptides have side chains attached to the α-carbon and peptoids have side chains attached to the amide nitrogen. Peptoids can be synthesized using an automated peptide synthesizer, and they are more cost efficient than a peptide because the backbone amines do not have to be protected. The method for synthesizing peptoids is carried out by a submonomer solid-phase protocol shown in Figure 2. This works by having two submonomers that are used to assemble the N-substituted glycines (NSG) monomers.4 Peptoids are synthesized to include a unique set of side chains by utilizing any free amine. Peptoids also possess helices that are robust and stable. 2 Figure 1: Comparison of peptide and peptoid backbone Figure 2: Submonomer solid-phase protocol scheme It has also been found that properly designed peptoids have the ability to be inexpensive and efficient protein ligands. They have the ability to not only bind efficiently with an unmodified protein but to bind specifically in the presence of other bacterial proteins.1 These facts about peptoids make them an ideal candidate for affinity based chromatography techniques. The goal of this study was to evaluate the potential of peptoids to bind with protein. Methods and Materials Preliminary Binding Experiments The first test of the potential of peptoids to bind to protein was performed using an unknown peptoid sequence and cell lysate. The peptoid was synthesized previously on amide resin. The experiments were performed with the peptoid attached to the resin. The cell lysate was prepared by sonicating the mixture to break the cell membranes, and the solution was then centrifuged to remove the cell debris. The cell lysate was diluted 4-fold to a final sample volume of 1mL in 3 different buffers: Phosphate Buffer Solution (PBS), 0.02M Na2HPO4 1M Na2SO4(HIC-A) and 0.02M Na2HPO4 (HIC-B). Then 500μL of each protein sample was added to the peptoid resin and incubated for 72 hours at room temperature while being gently shook. A bicinchoninic acid(BCA) assay was performed to determine the protein concentrations of the samples before and after the incubation period with the peptoid resin. The BCA assay was completed by combing 20 μL of the protein sample , 200 μL of reagent A (mixture containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide), and 5 μL of reagent B(containing 4% cupric sulfate) in a 96-well plate. The samples were then incubated for 30 minutes at 37°C. The assays produce a color change dependent on the amount of protein in the sample. The protein concentrations were then determined by using a nanodrop spectrophotometer at a wavelength of 562nm. A bovine serum albumin (BSA) standard curve was also developed using this method. Preliminary Validation A series of validation steps were executed to determine whether the protein was actually binding with the peptoid. First, the protein sample was removed from the sample. Then a high salt concentration solution, 1M NaCl, was added to the peptoid resin in order to break the binding between the peptoid and the protein. Then a SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was done using a 12.5% acrylamide gel at 250 volts, 400 mA for 35 minutes. This was to show the presence of protein in the sample after the high salt wash. The next step was to liberate the peptoid from the resin. This was done using triflouroacetic acid (TFA) solution. After cleaving the peptoid from the resin another SDS-PAGE was done on the sample using a 12.5% acrylamide gel at 250 volts, 400 mA for 35 minutes. Peptoid Synthesis A peptoid that had previously shown protein binding ability was synthesized to further determine the binding potential.1 The peptoid was synthesized using Zuckerman’s sub-monomer protocol on amide resin.4 Each side chain was prepared with dimethyl formamide (DMF) as a solvent except for 4-(2-aminoethyl) benzene sulfonamide (Nbsa) which utilized dimethyl sulfoxide (DMSO). 7mL of a 1M solution was prepared. The peptoid sequence can be shown in Figure 3. Matrixassisted laser desorption/ionization (MALDI) was used to verify the successful synthesis of the peptoid. MALDI is a powerful mass spectrometry technique that will show the molecular weight of molecules present in the sample. Figure 3: Peptoid sequence utilized with a molecular weight of 1428 Binding Experiments Table 1 shows a summary of the binding conditions used in the final binding study. The main variations between different samples were the temperature and the protein concentration. Protein 1 had a concentration of 1.72mg/ml and protein 2 had a concentration of 0.663mg/mL. The two temperatures represent room temperature and a standard refrigerator. Each sample was incubated with 112 μL of protein solution. The mass of the peptoid resin was varied as stated. Table 1: Protein incubation conditions Sample Resin Mass Protein 1 Protein 2 22° C 4° C