Patent Application
20060172282
This invention relates in general to a peptides that can bind to inorganic surfaces (metals, metal oxides and semiconductors) and other nanometric structures such as carbon nanotubes and that can thus be used to serve as templates to control the growth and nucleation of inorganic nanoparticles in vitro. These peptides include ones that can bind to diverse materials that range from metals such as silver, gold, platinum and cobalt nanoparticles, inorganic metal oxides such as silica, cobalt oxide, iron oxide, zinc oxide and tin oxide, semiconductor materials such as palladium, gadolinium, and germania to other materials such as ruby, carbon nanotubes and sodium montmorillonite, and they are preferably obtained from a phage display library using a polymerase chain reaction (PCR)-driven method. The peptides of the invention are also capable of functioning as templates for the synthesis of silver and cobalt platinum nanoparticles.
Biomineralization is a widespread phenomenon in nature wherein many biological systems are capable of forming structures from varied inorganic substrates. Biomolecules which have the properties of binding inorganic structures such as metals and metal oxides will be particularly important in applications on the nanometric scale. In addition, biomolecules will can extremely useful in the future because they can act as templates whereby due to their particularly properties, and thus they can be used to arrange and organize inorganic substances from the nanoparticle level and up. For example, silver, magnetite, and cadmium sulfide particles can be microbially produced, and marine organisms such as diatoms and sponges are known to synthesize siliceous structures. In general, the transformation of inorganic molecules into nano- and microstrutured components on the biological scale appears to be controlled by proteins.
Nanoparticles and nanomaterials are highly desirable in many applications because these materials have unique optical, electronic and magnetic properties that arise due to their quantum size confinement. As indicated above, there is an emerging field of nanobiotechnology which seeks to employ the use of biomolecules as templates for the synthesis of nanomaterials. Natural biological systems, which master ambient conditions chemistry, often can synthesize inorganic materials that are hierarchically organized from the nano- to the macro-scale. Numerous microorganisms are capable of synthesizing inorganic-based structures, for example, microorganisms can synthesize iron oxide, silica, silver, gold and cadmium sulfide nanoparticles.
However, the use of proteins and other biomaterials from such microorganisms to possibly direct the assembly of nanostructured components into sophisticated functional structures has remained hard if not impossible to accomplish efficiently and has long been a desired goal. The ability to utilize proteins to produce nanostructured inorganic materials, such as those made of silica or other similar compounds in vivo under ambient conditions would provide a significant advantage over traditional approaches to materials synthesis which require stringent conditions such as high temperature, pressure and pH. It is thus highly desirable to be able to produce nanometric metallic or other inorganic materials using a biomimetic approach which will only require ambient conditions to produce useful inorganic structures such as those useful on a nanometric scale with a minimum amount of complexity and expense.
In the biological arts, it has long been known to utilize a phage display library to express a particular protein. Phage peptide display is a selection technique in which random peptides from a library are expressed as a fusion with a phage coat protein, resulting in the display of the fused protein on the surface of the phage particle. The advantage of phage display technology is that it can offer the ability to identify surface-specific proteins in a more practical way and avoid the lengthy and complex identification procedures associated with traditional protein isolation and gene sequencing. However, it has not previously been known to utilize phage display technology in such a manner as to identify and produce peptides which can exhibit binding and nucleation properties against an inorganic material such as silica, silver, cobalt, iron, etc., in order to direct the precipitation of these materials so as to be able to create useful structures on a nanometer scale. Accordingly, it is thus highly desirable to develop a method for utilizing phage display libraries in order to allow rapid selection of surface-specific peptides and identify a subpopulation of silica-precipitating peptides, or peptides that can be used to catalyze the precipitation or deposition of other inorganic materials, and to use such peptides as templates for “bottom-up” microfabrication.
In addition, there are numerous applications wherein the delivery or removal of inorganic agents plays an important role in the complexity, expense and efficiency of the particular method. For example, in the case of toxic waste areas, it is very often the case that the most toxic ingredients that need to be removed from a site are the heavy metals which are extremely toxic and sometimes even radioactive. In the case of the infamous Love Canal site, metals in toxic levels discovered at the site included aluminum, antimony, beryllium, cadmium, copper, iron, lead, selenium, silver and zinc. At present, although there are many known methods for attempting to remove toxic levels of metals at such a site, many of these methods are often expensive, inefficient and general in nature and thus may not be adequate to eliminate or reduce levels of particular metals. Even further, it is important to be able to clean up and contain radioactive waste which may also be present at such sites, and once again it is desirable to achieve a method and product whereby such radioactive materials can be specifically bound and removed when necessary.
The use of phage peptide display libraries to select peptides that bind to inorganic surfaces has been disclosed, for example, in PCT Published Application WO 03/078,451, incorporated herein by reference. However, this system involved the labor-intensive procedures of phage amplification which would result in slowing down the process and in making it more expensive and cumbersome.
It is thus still remains desirable to utilize these methods and develop peptides which can be used as templates in order to promote synthesis of inorganic or organic materials on a nanometric scale, but with improved efficiency so that it can be conducted on a larger yet less expensive scale.
Accordingly, it is thus an object of the present invention to provide peptides that can bind to inorganic surfaces (metals, metal oxides and semiconductors) or other objects (e.g., carbon nanotubes) on a nanometric scale and which can thus be used to serve as templates to control the growth and nucleation of inorganic nanoparticles in vitro.
It is further an object of the present invention to provide a system wherein a phage display peptide library is used in the identification and isolation of peptides which can bind silica or other nanometric materials and which can thus be useful as templates in methods of fabricating structures on an nanometric scale.
It is still further an object of the present invention to isolate and identify useful peptides which can bind to inorganic or organic materials using a method that is quick, efficient, and which can be carried out with a minimum of steps and without the need for rigorous physical conditions.
It is still further an object of the present invention to develop and provide peptides which can be used to catalyze the precipitation and deposition of useful inorganic materials such as silica, silver, germanium, cobalt oxide, iron oxide and other metals and metal oxides.
It is yet a further object of the present invention to develop and provide peptides which can be used as templates to direct the development of many functional nanometric materials such as carbon nanotubes.
It is even further an object of the present invention to develop and provide peptides which can be used as templates for the synthesis of nanoparticles from inorganic materials such as silver, gold, platinum, cobalt, silica, iron, zinc, tin, palladium, gadolinium, germanium, and oxides thereof.
It is yet further an object of the present invention to isolate and identify useful peptides which can bind to potentially toxic inorganic materials and thus be used in methods of delivering or removing said materials when necessary.
It is yet further an object of the present invention to develop and provide peptides which can be used to remove or delivery radioactive materials in an efficient and relatively inexpensive manner.
These and other objects are achieved by virtue of the present invention which provides a method for identifying and isolating peptides capable of binding to inorganic materials and other nanometric particles such as carbon nanotubes using a combinatorial phage display peptide library and a step involving the polymerase-chain reaction (PCR). By the present method, it is possible to eliminate the labor-intensive and inefficient procedures of phage amplification as used in prior art methods and directly obtain sequence information of interacting peptides in a single step using the PCR method. In the method in accordance with the invention, a combinatorial phage display library is used to isolate and select the desired binding peptides by a series of steps of target binding, elution and amplification which may be repeated until the desired amount of phage expressing peptides with the desired binding properties is obtained. Once these phage are isolated and/or purified following this procedure, the phage coating is ruptured or otherwise removed so as to release the phage nucleic acids, and a step involving the polymerase-chain reaction is utilized in order to obtain the sequences of the peptides binding to the particular nanoparticles introduced to the phage display library. Once identified in this manner, the peptides then may be expressed and used as templates to guide the precipitation and synthesis of useful structures on a nanometric scale.
These and other features of the present invention as set forth in, or will become obvious from, the detailed description of the preferred embodiments provided hereinbelow.
In accordance with the present invention, there is provided a method for utilizing a combinatorial phage display library to identify and obtain peptides which can bind to nanometric particles including organic materials such as carbon nanotubes and inorganic materials such as silica and other metals and metal oxides and which can be used to catalyze the precipitation and deposition of those materials on a nano/micrometer scale. In the preferred process, the nanometric material-binding peptides are obtained using a suitable combinatorial phage display peptide library such as would be commercially available and well known in the art. Phage peptide display is a selection technique in which a library of random peptides are expressed as a fusion with a phage coat protein resulting in the display of the fused protein on the surface of the phage particle. In the present invention, a suitable combinatorial library will be one in which phage expressing peptides binding to metals, metal oxides, and other inorganic and organic materials on the nanometric scale can be identified. One such combinatorial phage display library is the 12 amino acid phage peptide display library (PhD-12) was purchased from New England Biolabs, Inc (Beverly, Mass.). The phage-display peptide library consists of 109 different phage clones, each displaying a unique 12 amino acid peptide on the phage surface.
In the general process of the invention, as explained further below, the nanometric material-binding peptides of the invention are obtained by first incubating the target particles with the combinatorial phage display peptide library. As indicated below, target particles in accordance with the invention will include any suitable inorganic or organic material (e.g., carbon nanotubes) that can be bound by peptides and which can be deposited or precipitated to form an appropriate nanostructure. Examples of such inorganic materials include metals and metal oxides currently used in applications on a nanometric scale including silver, gold, platinum, cobalt, silica, iron, zinc, tin, palladium, gadolinium, germanium, and oxides thereof. In addition, it is also possible to bind other inorganic materials which may be important in methods of removing or delivering these metals or oxides for various purposes. Accordingly, inorganic materials that can be removed using the process of the invention include aluminum, antimony, beryllium, cadmium, copper, iron, lead, selenium, silver and zinc. Still other materials such as those which would be potentially recoverable from aqueous environments such as lakes, streams, etc., and these would include gold, platinum, palladium, and oxides thereof. Further, it is also desired to develop means of either removing or delivering radioactive metals, such as uranium, radioactive cobalt, etc., and thus these metals as well can be bound using the peptides prepared in accordance with the process of the invention, as explained further below. In short, the diverse nanometric materials that can be utilized in conjunction with the present method range from metals such as silver, gold, platinum and cobalt nanoparticles, inorganic metal oxides such as silica, cobalt oxide, iron oxide, zinc oxide and tin oxide, semiconductor materials such as palladium, gadolinium, and germania to other materials such as ruby, carbon nanotubes and Na+ montmorillonite.
The present invention thus can be used to obtain peptides that will specifically bind with all of the inorganic elements conventionally known, where stable in the pure form, such as would be reflected in the Periodic table of elements. It is also contemplated that the present method will be useful to isolate and/or identify peptides which can bind to these inorganic elements along with their stable inorganic complexes as well, e.g., oxides, etc. of these elements.
In the preferred process, as explained further below, the phages from the combinatorial library are identified and sequenced using a PCR process and selected for their ability to express peptides that exhibit selective affinity for a particular nanometric material and which will be able to guide the deposition and precipitation of that material such as in the form of a template for nanometric structures. In the preferred process in accordance with the invention, peptides which can bind to an inorganic material or other nanometric materials such as carbon nanotubes can be isolated and identified via the steps of incubating a combinatorial phage peptide display library with a target material for a time sufficient so that the nanometric material will bind to peptides expressed by the phage of the library; eluting the library so as to collect the phage bound to the target material; rupturing the phage so as to release the nucleic acid of the phage bound to the target material; amplifying the nucleic acid of the phage bound to the target material using a polymerase-chain reaction (PCR); and sequencing the phage nucleic acid so as to determine the sequence of the peptides coded by the nucleic acid of the phage which can bind to the target nanometric material.
As indicated above, in accordance with the present invention, it is preferred that a combinatorial phage display peptide library such as the 12 amino acid phage peptide display library (PhD-12) be utilized in the invention, however, other available amino acid libraries having suitable peptides of other lengths would also be useful in the invention. In the preferred process, the phage display library is incubated with the desired target material as described above so as to target phage which express peptides capable of binding with that material. Accordingly, the invention includes a step of isolating the desired phage such as by eluting the phage bound to the target and separating and collecting the desired phage. The use of the target particle to identify and isolate phage which express the peptides with the desired binding properties is known as “panning” or “biopanning”, and in the preferred process, multiple rounds of panning may be carried out as desired to further purify the selected phage and, increase the likelihood that the eluted and isolated phage will bind specifically to the target material. Ideally, the target particle is first itself isolated and purified before being used in the present process, such as through an acid wash or other suitable purification process.
In the preferred process of the invention, the method provides steps which go beyond the “panning” process and allow for the identification and isolation of many more binding peptides than was possible using previous methods. In this process wherein peptides are isolated via incubation of a phage peptide display library with a target nanometric material for a time sufficient to allow binding of the material to the peptides expressed by the phage of the library, the incubation step may be conducted using an appropriate phage display library (e.g., the Ph.D.12 library described above) and the desired nanometric material in a medium (e.g., a Tris-buffered saline solution containing 0.5% Tween 20, or TBST) for a time (e.g., 1 hour) and at a temperature (e.g., room temperature) suitable to allow the target material to bind with the peptides of the phage. This incubation step may optionally be followed by a step of washing the phages with a suitable buffer solution in order to assist in eliminating phages that do not specifically bind to the target material. Following the wash step, the phages of the library are eluted to collect the phage bound to the target material via a suitable elution step, e.g., through use of a suitable elutant such as 0.2 M glycine-HCl (pH 2.2) for a suitable time (e.g., 20 minutes).
The next step of the preferred process is to rupture the phage so as to release the nucleic acid of the phage bound to the target material, and this can be done in a number of suitable ways well known in the art. For example, the bound phage can be treated with a lysis solution (such as lysis buffer A comprised of 10 mM Tris-HCl pH 8.3, 10 mM ethylenediaminetetraacetic acid (EDTA), and 1% Triton X-100) for a time and at a temperature (e.g., 10 minutes at 95° C.) suitable to allow rupture of the phage coat and release of the phage DNA. At this point, the nucleic acid of the phage binding peptides can be sequenced using the techniques described, for example, in PCT application WO 01/79479, incorporated herein by reference. In the preferred process, once the desired phage expressing the peptides are isolated and/or purified as described above, the nucleic acid (e.g., DNA) from the selected phages are then isolated and sequenced to obtain the genetic information encoding for the displayed peptides and determine the sequence of the peptides coded by the nucleic acid of the phage which can bind to the target nanometric material. This may occur through a number of suitable techniques well known in the art including amplification of the genetic material by known processes such as an automated sequencer or other suitable PCR techniques.
In the particularly preferred process, such as those recommended by the manufacturer, a suitable amount of the PCR materials (e.g., 50 μl of PCR REDTaq ReadyMix PCR master mix manufactured by Sigma-Aldrich, St. Louis, Mo.) was directly added to the tube containing the lysed peptide displaying phages. The phage DNA sequences in this process can be amplified such as by adding to the tube 1 μl each of forward (5′-CCTCGAAAGCAAGCTGATMC-3′) and reverse (5′-GTACCGTAACACTGAGTTTCG-3′) primers. The PCR amplification can be performed for a suitable time, e.g., using 20-25 cycles of denaturation at 95° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 72° C. for 30 sec, followed by a final extension cycle of 72° C. for 5 min. The PCR products can then be separated such as on a 1.2% agarose gel by DNA electrophoresis. The PCR products are then preferably cloned into the TOPO vector (Invitrogen, San Diego, Calif.) according to manufacturer's instructions. The clones obtained in the present invention can then be sequenced using an automated DNA sequencer using standard sequencing methods.
As indicated above, the process of the invention can be carried out on any desired nanometric material, such as an inorganic metal or metal oxide or other useful nanometric structure such as carbon nanotubes, that may be used in applications where such materials may be deposited or precipitated on a nanometric scale to form suitable structures such as those used in sensor arrays, microchips, etc. Among the inorganic materials useful in the invention are silica, silver, germanium, cobalt, iron, and the oxides of these metals and those other inorganic materials described above. Accordingly, the present invention includes the production or isolation and identification of those phage expressing peptides which bind to the target nanometric materials as well as to the amino acid sequences of the expressed peptides and the nucleic acid sequences encoding said amino acids. Examples of suitable peptides that can be obtained in accordance with the invention are those shown in
As indicated above, these peptides may be expressed and utilized in a number of suitable applications as would be understood by one skilled in the art, but in particular, the nanometric material-binding peptides obtained by virtue of the present invention are ideally used as templates in nanometric material synthesis and may be used to guide the development of important microstructures on a nanometric scale such as would be used in sensors, computer chips and the like. In the preferred embodiments, the peptides obtained in accordance with the invention will be suitable for catalyzing and promoting the precipitation or directing growth of the particular inorganic material which is the target of the peptide of the invention.
In addition to the embodiments described above, the present invention may be used in a number of beneficial applications. For example, it is also possible to bind other inorganic materials which may be important in methods of removing or delivering these metals or oxides for various purposes. Accordingly, the present invention contemplates a method for recovering an inorganic material using a peptide identified and/or isolated in accordance with the invention as set forth above, by introducing an amount of the said peptide to the area or site where recovery or elimination of the particular inorganic material is desired, maintaining said peptide at said site for a time effective to achieve the desired level of peptide binding to the inorganic material, and then removing the bound peptide so as to recover or eliminate the particular metal bound by the peptide. In these applications, the effective amount of the peptide will vary depending on the type of application, and one skilled in the art would appreciate that each individual job would have an appropriate amount of peptide depending on the circumstances of the application.
For example, the peptides of the invention can be used in cases wherein recovery of a valuable inorganic material is desired, such as the mining of metals such as silver, gold or platinum from lakes and streams. In these applications, a number of suitable ways could be used to carry out such removal, including use of synthesized versions of the binding peptides identified from the above process, or via a recombinant genetic vector, such as an bacterial organism using a plasmid or viral vector with the genetic instructions to express the peptides in accordance with the invention. One suitable vector would be to have the peptides expressed in E. coli which could be prepared in suitable amounts, introduced to the body of water, whether natural stream or lake or artificial enclosure such as a tank or vat, given suitable time for the expression of the peptides and the binding to the precious metals, and then recovery or filtering of the peptides bound to the precious metals, following which the peptides could be separated through various means well known in the art. In addition, it is possible to prepare the peptides or E. coli expressing such peptides in a suitable vehicle, such as a filter or cartridge, e.g., where the peptides could be linked to a solid support (e.g., agarose, resins, polysaccharides, etc.) in such a way that their binding site is unaffected. Similar steps could be taken where the inorganic material is a toxic product, e.g., toxic metal waste or radioactive waste, and in these cases, the recovery of the metal in this procedure would be followed by its disposal or containment in a suitable manner. In addition, such peptides could be used in chelation methods of eliminating targeted inorganic materials from a human or animal patient.
Similarly, the present invention contemplates applications wherein delivery of a particular inorganic material is highly desirable, such as the direct application of a radioactive material, such as radioactive cobalt, to a tumorous cell in a cancer patient. In general, the method of delivering an inorganic material bound to a peptide identified and/or isolated in accordance with the invention as set forth above, can be carried out binding the peptide to the desired inorganic agent, and linking the bound peptide to a means of delivering the bound peptide to a particular site. For example, in the case of cancer treatment, it is possible to link the peptide to an antibody that can target particular tissues, such as cancerous tissues or cells, and the antibody-peptide-bound inorganic material complex can be introduced in a patient where it will apply the necessary agent, e.g., radioactive cobalt, directly to the site of the tumor. In such a case, the peptide will be appropriately linked to the antibody in such a manner that the binding property of the peptide to the inorganic material is unaffected and the target binding site of the antibody is unaffected.
Still other applications of the peptides of the invention would be in an area or site where recovery or elimination of the particular inorganic material is desired, maintaining said peptide at said site for a time effective to achieve the desired level of peptide binding to the inorganic material, and then removing the bound peptide so as to recover or eliminate the particular metal bound by the peptide. In these applications, the effective amount of the peptide will vary depending on the type of application, and one skilled in the art would appreciate that each individual job would have an appropriate amount of peptide depending on the circumstances of the application. Such a method will be useful in applications such as recovery or valuable metals or elimination of toxic inorganic materials. Another application of specific high binding peptides in accordance with the invention is that they can be directed to metals applied in NMR and X-ray contrast agents (e.g., as carried by the fragment or fused to a larger protein).
In accordance with the invention, a method can also be utilized for obtaining phage which can express the peptides of the invention. In such methods, the phage is obtained in a method comprising the steps of incubating a combinatorial phage display peptide library with a target inorganic material which will bind to peptides expressed by the phage of the library; eluting the library so as to collect the phage bound to the target inorganic material; and amplifying the nucleic acid of the phage bound to the target inorganic material using a polymerase-chain reaction (PCR) and sequencing said nucleic acid so as to determine the sequence of the peptides coded by the nucleic acid of the phage which can bind to the target inorganic material.
In addition, the present method can be used to identify peptides which can bind to a stable inorganic element or a stable inorganic complex of that element using a combinatorial phage display library as described above. In such a method, these peptides are identified by the steps of incubating a combinatorial phage display peptide library with a target inorganic element or complex which will bind to peptides expressed by the phage of the library; eluting the library so as to collect the phage bound to the target inorganic element or complex; isolating the nucleic acid of the phage bound to the target inorganic element or complex; amplifying the nucleic acid of the phage bound to the target inorganic element or complex using a polymerase-chain reaction (PCR); and sequencing the nucleic acid so as to determine the sequence of the peptides coded by the nucleic acid of the phage which can bind to the target inorganic element or complex.
It is thus submitted that the foregoing embodiments are only illustrative of the claimed invention and not limiting of the invention in any way, and alternative embodiments that would be obvious to one skilled in the art not specifically set forth above also fall within the scope of the claims.
The following examples are presented as illustrative of the present invention or methods of carrying out the invention, and are not restrictive or limiting of the scope of the invention in any manner.
Overview
Phage peptide display libraries are commonly used to select for peptides that bind to inorganic surfaces (metals, metal oxides and semiconductors). These binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles in vitro. In this report, we describe the identification of a unique set of sequences that bind to silver and cobalt nanoparticles from a phage peptide display library using a Polymerase Chain Reaction (PCR)-driven method. The amino acid sequences obtained by the PCR method are a distinct set of sequences that would otherwise be missed using the regular panning method. Peptides identified by the method described here are also shown to function as templates for the synthesis of silver and cobalt nanoparticles.
Background
Nanomaterials have unique optical, electronic and magnetic properties that arise due to their quantum size confinement. Nanobiotechnology, an emerging discipline, seeks to employ the use of biomolecules as templates for the synthesis of nanomaterials. Biological systems, a master of ambient conditions chemistry, synthesize inorganic materials that are hierarchically organized from the nano- to the macro-scale. Numerous microorganisms are capable of synthesizing inorganic-based structures, for example, microorganisms can synthesize iron oxide, silica, silver, gold and cadmium sulfide nanoparticles.1-7 The process of biomineralization and assembly of nanostructured inorganic components into hierarchical structures has led to the development of a variety of approaches that mimic the recognition and nucleation capabilities found in biomolecules for inorganic material synthesis. A number of studies have demonstrated that proteins identified from biological organisms can be used as catalysts or templates for material synthesis in vitro.8-11
These proteins control the nucleation and growth of the inorganic structure. Amino acids are known to interact with metal ions, for example, histidine, arginine, lysine, cysteine, methionine, tryptophan, aspartate, and glutamate are known to form complexes with metal ions.12-15 The presence of a particular sequence of amino acids within a peptide provides the molecular recognition motif required to interact with a given target (metals, metal oxides, semiconductors). Random peptide libraries are screened to select or evolve ligands with specific amino acid sequences that recognize the inorganic surfaces.16-23 This approach of “evolution in a test tube” allows for the selection of amino acid sequences that exhibit high affinity for inorganic surfaces. In some cases, the specific molecular recognition properties of selected peptides provided by the amino acid functional groups can be used to control the nucleation and growth of the material it was selected against.
Phage peptide display is a powerful technique for selecting peptides with novel properties.24 In phage peptide display, a combinatorial library of random peptides (˜109) is usually expressed as a fusion protein with the phage minor coat protein (pill). As a result, the peptide is displayed on the outer surface of the phage particle with each phage particle displaying 5 copies of the same 12 or 7 amino acid peptide sequence. Since the DNA sequence for the displayed peptide is genetically fused to the pill gene, recovery of the phage DNA allows for deciphering the amino acid sequence of the selected peptide. The procedure, also know as “panning”, is carried out by incubating the library of phage displayed peptides with a target, washing away unbound/non-specific phages, and eluting the bound phage using a low pH incubation step. The eluted phage are amplified and subjected to additional panning rounds to evolve highly enriched binding sequences. Finally, the DNA from individual phage clones is isolated and sequenced. We have observed that a number of phage displayed peptides remain strongly bound to the target even after an extended acid elution step. Since these strong binding sequences are essentially lost during the regular panning method, many peptides with the highest affinity for the target are potentially missed. If the goal is to use these peptides as templates for the nucleation and growth of materials that they were selected against, then a unique peptide set has been overlooked in work to date. In this report, we show the rapid identification of peptides that interact with silver and cobalt nanoparticles and demonstrate that these peptides are efficient templates in the synthesis of silver and cobalt nanoparticles.
In an earlier study, we identified three peptides from the phage peptide display library that interacted with silver nanoparticles using the regular acid elution-based panning method.22 Only one out of the three silver binding peptides was capable of efficient silver nanoparticle synthesis. This peptide, AG4, reduced silver ions to metallic silver without the need of an external reducing agent.22 The identification of only three binding sequences from the combinatorial library prompted us to confirm whether all the peptide displaying phages were eluted from the surface of the silver nanoparticles.
Experimental Procedures
PHAGE PEPTIDE DISPLAY SCREENING: Silver and cobalt binding peptides were selected using the Ph.D.-12 phage display peptide library obtained from New England Biolabs, Inc (Beverly, Mass.). The target binding, elution and amplification were carried out according to manufacturer's instructions. Briefly, the peptide library was incubated with acid washed silver or cobalt nanoparticles (Sigma-Aldrich, St Louis, Mo.) in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 1 hr at room temperature, followed by several washes in TBST buffer (0.5%-0.8% Tween 20). The phages were eluted from the particles by the addition of 0.2 M glycine-HCl (pH 2.2) for 20 minutes. The eluted phage were then transferred to a fresh tube and neutralized with Tris-HCl, pH 9.1. The eluted phage were then tittered and subjected to 3 additional pannings (Set I). After the final panning procedure, Esherichia coli ER2537 host cells were infected with the eluted phage and plated on Luria Broth (LB) plates containing X-Gal and IPTG. DNA was isolated from 30 independent blue plaques and sequenced using an ABI 310 (PE Applied Biosystems, CA) automated sequencer.
PCR METHOD: The PCR method was performed as described with some minor modifications25. Binding of the phage peptide library with the nanoparticles was done as described above. The phages were either washed several times in TBST buffer only (Set II) or washed in TBST buffer followed by elution with 0.2 M glycine-HCl pH 2.2 (Set I). The phage-nanoparticle complex was incubated in 48 μl lysis buffer A (10 mM Tris-HCl pH 8.3, 10 mM EDTA, 1% Triton X-100) for 10 min at 95° C. 50 μl PCR REDTaq ReadyMix PCR master mix (Sigma-Aldrich, St. Louis, Mo.) was directly added to the tube containing the lysed peptide displaying phages. The phage DNA sequences were amplified by adding to the tube 1 μl each of forward (5′-CCTCGAAAGCMGCTGATAAC-3′) and reverse (5′-GTACCGTAACACTGAGTTTCG-3′) primers. The PCR amplification was performed using 20-25 cycles of denaturation at 95° C. for 30 sec, annealing at 50° C. for 30 sec, and extension at 72° C. for 30 sec, followed by a final extension cycle of 72° C. for 5 min. The PCR products were first separated on a 1.2% agarose gel by DNA electrophoresis. The PCR products were cloned into the TOPO vector (Invitrogen, San Diego, Calif.) according to manufacturer's instructions. The clones were then sequenced using an automated DNA sequencer using standard sequencing methods.
TRANSMISSION ELECTRON MICROSCOPY (TEM), ENERGY DISPERSIVE X-RAY ANALYSIS (EDX) AND ELECTRON DIFFRACTION: The washed particles were mounted on carbon-coated copper grids. Micrographs were obtained using a Philips FEG200 operating at 200 kV. EDX spectra were obtained using a Noran Voyager system attached to the TEM. Electron diffraction for single crystals was also obtained on the Philips TEM.
Results and Discussion
We adapted a polymerase chain reaction (PCR) method25 to detect for the presence of peptide displaying phage DNA on the silver nanoparticles using a specific set of DNA primers. If some of the peptide displaying phages were resistant to acid elution and remain bound to the nanoparticles, then the presence of a phage DNA fragment after the PCR reaction indicates the presence of phage DNA. The DNA primers used for PCR amplified a 0.33 kilobase (kb) region of the phage DNA including the region encoding for the displayed peptide (see supplemental information). The silver nanoparticles are incubated in lysis buffer A (100 mM Tris-HCl, pH 8.2, 10 mM EDTA, 1% Triton X-100) and heated to 95° C. for 10 min to disrupt the phage coat resulting in phage DNA release.
The PCR reaction mix was directly added to the silver nanoparticles and placed in a thermocycler for PCR amplification. Following the PCR reaction, the samples were separated on a 1.2% agarose gel (
The specificity of the PCR reaction was indicated by the absence of an amplified DNA fragment when either one of the two DNA primers is omitted in the reaction (
The PCR method was also used to identify peptides that remain bound to cobalt nanoparticles. However, in this case we decided to investigate in detail the utility of the PCR method as an alternative to the traditional panning approach. In order to address this issue, we compared the peptide sequences obtained before elution and after acid elution to the peptide sequences obtained after 4 rounds of regular panning. Three different sets of cobalt nanoparticles were incubated with the phage peptide display library. After several buffer washes, one set was acid eluted with 0.2 M glycine-HCl, pH 2.2 for 20 min and then used for the PCR amplification reaction (Set 1), the second set was only washed several times in buffer and then subjected to PCR amplification (Set II) and the third set underwent the traditional 4 rounds of panning (Set III). The DNA agarose gel electrophoresis of the PCR reaction from both the eluted and uneluted cobalt nanoparticles revealed the presence of the 0.33 kb diagnostic phage DNA fragment (
The PCR fragments from both the sets were cloned and sequenced. The amino acid sequences of the cobalt binding peptides are listed in Table 2. As expected, the sequences of the uneluted nanoparticles (Set II) contained three groups of sequences, one group that was the same set of sequences as that from the eluted nanoparticles (Set I), a second set that was similar to the sequences obtained from the regular panning (Set III), and a third sequence set that was absent from either of the other two groups. One would expect that Set II sequences would represent all of the sequences contained within Set I and Set III; however, seven of the sequences did not belong to either Set I or Set III. This can be explained by the fact that a larger number of clones would have to be sequenced in order to fully sample the diversity set. But more importantly, the sequence data clearly showed that a large amino acid sequence space was being lost by the regular panning method. Over 35 independent peptide displaying phages from the regular panning method were sequenced—resulting in the selection of 4 different amino acid sequences that bind to cobalt nanoparticles by the regular panning method. This confirmed that the PCR method identified a unique set of peptide sequences that were not recovered by the regular panning techniques.
Based on the sequences obtained in Set II, it is possible to eliminate the labor-intensive procedures of phage amplification and directly obtain sequence information of interacting peptides in a single step using the PCR method. Analysis of both the silver and cobalt nanoparticle-binding peptides revealed some interesting sequence characteristics.
For example, all the silver binding sequences obtained by the regular panning methods lacked the amino acid cysteine (C). Cysteine is a thiol-containing amino acid that binds to silver and gold ions. Four of the sequences obtained by the PCR method contained cysteine. Furthermore, the amino acids glutamic acid (E), histidine (H), isoleucine (I), and tryptophan (W) were present in the sequences obtained by the PCR method but were absent in all of the sequences obtained by regular panning. For the cobalt binding peptides, the sequences obtained by regular panning lacked the amino acids cysteine (C), glutamic acid (E), histidine (H), isoleucine (I), tryptophan (W) and tyrosine (Y). It was surprising that these sequences lacked histidine and glutamic acid—amino acids that are known to bind to cobalt.12-14 It is plausible that our high stringency wash conditions used in selecting the cobalt binding sequences eliminated amino acid sequences that contained these amino acids. We believe that this is unlikely since peptide sequences in Set II, obtained using the same stringency wash conditions, contained histidine residues. An alternative explanation could be that during successive rounds of panning, the histidine containing sequences were diluted out by the dominant stronger-binding sequences that were identified in the fourth round.
In order to address whether some of the cobalt binding sequences identified in Set I or II would function as templates in the synthesis of cobalt-platinum (CoPt) nanoparticles, the synthesis of CoPt nanoparticles was performed in the presence of the cobalt binding peptides. CoPt is a magnetic alloy and is a candidate for ultrahigh-density magnetic recording media because of its enhanced magnetic anisotropy and other properties.27 Random nucleation and growth of magnetic nanoparticles results in large sizes, and broad size distributions, which adversely affect the magnetic properties. It is possible that by using biological templates that nucleate metal ions, the random nanoparticle nucleation and growth process can be more controlled. Peptide (10 mg/ml) was added to a solution containing 1 mM ammonium tetrachloroplatinate and 1 mM cobalt acetate tetrahydrate in 0.1 M HEPES buffer pH 7.5. The solutions were mixed and stored for 4 hrs to overnight at 4° C. 10 μl of 25 mM sodium borohydride was then slowly added to the solution to reduce the metallic precursors. The addition of reductant resulted in the formation of a homogenous greenish solution (
Similarly, Co1-P1 as well as the peptides from the regular panning method all resulted in the precipitation of the metal alloy. Analysis of the Co1-P10 stabilized CoPt solution by transmission electron microscopy (TEM) revealed the presence of discrete nanoparticles with an average diameter of 3.5 nm±0.5 nm (
In conclusion, we have shown here the identification of biological templates that can be used in the nucleation and growth of inorganic nanoparticles using a PCR-based approach. The PCR-based approach of screening peptide-displaying phages allows for the rapid identification of strongly interacting amino acid sequences that would be likely absent from the sequences obtained via regular phage peptide display panning methods. Albeit, with either method, screening of the binding peptides will have to be done in order to obtain the desired properties of controlled nucleation and growth.
The following references cited in the text above are incorporated by reference as if set forth in full herein.
27) Hyeon, T. Chem. Comm. 2003, 1, 927-934.
