Shedding Light on Peptide Synthesis

BY JOHN MARK CARTER, STEVE V •ANALBERT, JAIME LEE, JEFF LYON, & CAROLYN DEAL

ne of the toughest method arc typically eight amino acids solutions overnight. problems encoun• long and overlap each other through• The major weakness of the peptide tered in vaccine out the entire protein sequence. For pin procedure is the complexity of si• development is example, if a certain protein antigen is multaneously synthesizing hundreds or delineating the antibody response to a composed of a string of one hundred even thousandsofpeptides. The "amino protein antigen. Whereas the overall amino acids, the first acid indexer" pre• response to an antigen may involve vari• peptide would com• sented here was de• ous antibody species, each antibody prise amino acids 1- signed to simplifY the molecule can bind specifically to only 8. The second one technical problems one unique epitope. Often, antibodies would comprise of matching the dif• to a small subset of these epitopes will amino acids 2-9, and ferent amino acids to block a protein's function, clearing in• so on, up to the 94th the hundreds of fectious organisms or provoking other peptide, which would microtiterplatewells processesinancffcctiveimmuneresponse. be 94-100. The pep• for each day's work Protein cpitopes can be reproduced by tides arc prepared on in a Geyscn peptide synthetic processes that duplicate the an 8 x 12 rectangular synthesis. We have protein'saminoacidsequence.Antibod• matrix of plastic pins developed computer ies often bind to these synthetic pep tides (Figure 1). Each pep• software and hard• Figure 1. Prederivatized peptide pins. much as they would bind to the intact tide chain is as• ware that allows the The polyethylene pins are arranged in protein antigen. One effective way to sembled onto one relatively straightfor• an 8 x 12 matrix on 9-mm centers (like analyze and map the binding specificity pin throughout the ward creation of up standard microliter plates) and are of an antibody is to make a number of synthesis. The pep• to 960 separate pep• commercially available from Cam• synthetic peptides and test them for anti• tides are synthesized tides (10 different bridge Research Biochemicals and ICI body reactivity in a standard capture from their C-termini microtiter plates) si• Bio Products & Fine Chemicals. ELISA. A simple method for making and according to well-de• multaneously testing large numbers of peptides has fined peptide synthe• (VanAlbcrt et a!., been developed by Mario Geysen ( Geysen sis chemistry. Each 1991). eta!., 1984). In this technique, numerous day for eight days, pcptides are synthesized simultaneously one amino acid is MACHINE on individual chemically derivatized plas• added to each grow• CHARACTERISTICS tic pins, and hundreds of these pins are ing peptide chain. The outside of the later assayed for antibody binding via a This is done by amino acid indexer modified ELISA. This technique has en• pipetting a solution is constructed of a joyed great success in mapping medi• containing an acti• tough acrylic poly• cally and scientifically significant vated derivative of mer. This material is epitopes on numerous proteins (e.g., the correct indi• fairly inexpensive, Geysen eta!., 1984; Stover eta!., 1990). vidual amino acid Figure 2. Amino acid indexer. The easily machined, and The peptidcs made using the Geysen into the appropriate prototype is capable of simultaneous resistant to attack by corresponding wells synthesis of 960 peptides on 10 the solvents used in The authors are at the Walter Reed of a solvent-resistant, microliter plates. the synthesis proce- Anny Institute of Research, Washing• 96-well microliter dure. Ten polyethyl• ton, DC. The views expressed here do plate. When all the wells are filled with ene microtitcr plates are mounted on not necessarily reflect those of the U.S. the correct amino acids, the pins are the top face of the instrument, and a govemment. placed into the plates to react in the high-brightness red LED is mounted in

Figure 3. Overlapping •peptides. The sequences of the first four peptides that would be board drives the LEDs. These circuit prepared from a hypothetical parent protein sequence are shown. boards mate through two 50-pin con• nectors. The 10 latch boards are chained SKIP=O together and connected to the main control board using a bus cable. Data is communicated from the computer to the indexer using RS-232 asynchronous I I I I I I A B c D E F G H I ..J K L M N 0 p communication protocol. Because the I microliter plate wells are arranged in I I an 8 by 12 matrix, standard 8-bitASCII I I I I code is used. A single 8-bit character represents the light pattern of each SKIP=1 row, each bit corresponding to a single 8-MER PEPTIDES CREATED well. If the bit has a value of 1, the SKIP=O corresponding LED will be off; if the bit SKIP=1: has a 0 value, the LED will be illumi• ABCDEFGH ABCDEF G H nated. For each amino acid, 120 ASCII BCD EFGHI CDEFGH I J characters are transmitted to the amino CDE FGHI J EFGHI J K L acid indexer: one character for each of DEFGHI JK G HI J K L MN 12 rows for each of 1 0 plates. The indexer is designed to increase each of the wells. To use it, a researcher ent laboratory lighting conditions (Fig• the accuracy and throughput of pep• snaps an empty polyethylene microtiter ure 2). tide pin synthesis. When a synthesis of a plate into place over the top of each On the inside, the indexer has three few hundred samples is performed with LED-containing plate. This second plate major components: the main control current techniques, even an experi• serves as a reaction vessel for the pep• circuit board, the latch circuit boards, enced technician can suffer from men• tide syntheses. When the LEDs in the and the LED circuit board. Each of the tal fatigue and confusion while trying to bottom plates are lit, they shine through 10 LED plate modules is composed of make certain that all the microtiterplate the bottom of the upper (reaction ves• an LED circuit board that accepts the wells are correctly identified and filled. sel) plate, easily visible in typical ambi- individual LEDs, and a latch circuit The indexer simplifies the synthesis of

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This program also performs ELISA peptides in the typical ELISA applica• analysis and simplifies the processing of tion of the pins. By eluting the bound ELISA data once they have been re• antibodies from the individual pep tides corded by the micro titer plate reader. It it is possible to affinity-purify small quan• finds the lowest 25 absorbance values tities of antibody. The amounts of anti• on each plate and subtracts either the body protein isolated from each pin by average of the lowest 25 or the absor• this technique are vanishingly small, bance value for the negative control, but sufficient to be detected by means whichever is lower, from each value on ofbinding to Western blots (J.M. Carter, the plate. The resulting data matrix is unpublished observations). then stored in a format suitable for Finally, pin and indexer technology import into a spreadsheet program for may also be adapted to other biological manipulation, labeling, and output. The and biochemical investigations that uti• COST EFFECTIVE final results of a typical peptide pin lize a microtiter plate and repeated, ELISA prepared with the use of the pseudo-random addressing, including NITROGEN SOURCE amino acid indexer are shown in Figure lymphocyte proliferation, receptor FOR INDUSTRIAL 4 on the facing page. binding, cytotoxicity, viral plaques, en• zyme kinetics, and other procedures FERMENTATION PINS AND INDEXERS: APPLICATIONS often performed in clinical chemistry As the peptide pin method becomes and microbiology laboratories. It seems more widely used, indexer modifica• reasonable to expect that, as this meth• tions will be needed in order to support odology becomes more widely used and novel and inventive applications of pin accepted, even more applications will technology. One new application may present themselves. I I I involve the construction of mime topes. These are artificial epitopes made of REFERENCES peptides containing natural and non• !.DiPasquale, A., Bray, A.M., Geysen, H.M., YEAST natural amino acids in non-native se• Val erio, R.M., and Wang, J. 1991. Pro• quences (Geysen et al., 1986a,b). ceedings of the sixteenth annual confer• ence on protein structure and conforma• Mime topes-promising candidates for tion. Lome, Australia. e FUNGI future vaccines (McGregor et al. , 2.Geysen, H.M. , Me leon, R.H., and 1990)-can attain conformations in Barteling,S:J. I984.Proc.Natl. A cad. Sci. USA assays with the same binding character• 81:3998. istics as naturally occurring conforma• 3.Geysen, H.M., Rodda, SJ, and Mason, T J 1986a. Synthetic peptides as antigens. Ciba e MICROFILTERED tional epitopes. It seems likely that Found. Symp. 119:130. mimetopes may also be able to elicit 4.Geysen, H.M., Rodda, SJ., and Mason, T J. e LOW SALT antibodies with affinities for naturally 1986b. Mol. Immunol. 213:709. occurring conformational and even 5.Hagblom, P., Segal, E., Bill yard, E., and So, M. 1985. Nature 15:156. e HIGH CONTENT non-protein (e.g., carbohydrate) OF AMINO ACIDS epitopes. 6.Jarboe, D.L., Cassels, FJ., Kodak, J.A., Nauss, J.L., Boedeker, E. C., Deal, C. D., A second approach utilizes the chemi• Carter, J.M., and Reid, R.H. 1992. j. e LIQUID FORM cal spacer built onto the pep tides. This lmmunol. (submitted) . is the non-peptide moiety that attaches 7. Karabatsas, M., Seldon, M.A. , Napier, M.A., - 68%DM the pep tides to the plastic support pins. and Geysen, H.M. 1991. Proceedings of Incorporation of an acid-labile amino the sixteenth annual conference on pro• e PRICE RANGE FROM acid sequence (Asp-Pro) at this posi• tein structure and conformation. Lome, USD 1,5-2,5 Australia. tion in the peptide facilitates acidolytic 8. McGregor, D., Triantafyllou, B., Tribbick, PER KILO cleavage from the pin after synthesis is C., and Ceysen, H.M. 1990. Proceedings completed. This results in the genera• of the fifteenth annual conference on e A WIDE RANGE OF tion of a large number of soluble pep• protein structure and function. Lorne, ALTERNATIVES tides, albeit in limited quantities. This Australia. technique has proven useful in studies 9. Stover, C.K., Marana, D.P., Carter, J.M., Roe, B.A.,Mardis, E., and Oaks, E.V.1990. CONTACT demonstrating T-lymphocyte epitope Infectionandlmmunity58:2076. specificity through mitogenesis assays 10.VanAlbert, S., Lee,J., Lyon,J.A., and Primex for free sample kit and (van der Zee et al., 1989;Jarboe et al., Carter, J.M. (1991) Amino acid indexer test growth report. 1992), as well as peptide analog synthe• for synthesis of Ceysen peptides. Patent sis applications (DiPasquale eta!., 1991). application #07 / 679,990 (pending). For further information contactCRACO, Inc., Another fairly simple variation is the Biomedical Division, 2519 Meredith Drive, PRIMEX A/5 use of proteins other than antibodies to Vienna, VA 22181-4037. probe the peptide pin arrays. This 1l.van der Zee, R., van Eden, W., Meloen, Phone: + 47 4-816655 method has proven useful in some struc• R.H.,Noordzij,A.,andvanEmbden,J.D.A. Fax: + 474-817 580 ture-function studies on biological re• 1989.tur.Jlmmunol. 19:43. P.O. Box 183 ceptor molecules (Wang et al., 1991 ; 12.Wang, J., DiPasquale, A., and Ceysen, H .M. 1991. Proceedings of the sixteenth N-4270 Aakrehamn Karabatsas et al., 1991). A fourth ex• annual conference on protein structure ample takes advantage of the and conformation. Lome, Australia. NORWAY reversibility of binding of antibodies to Write In No . 206 on Reader Service Card

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