In further embodiments, the subject invention provides the use of energy-dispersive X-ray spectroscopy to measure the presence of counterions in peptide fibers, wherein the rate of annealing by irradiation is measured as a function of counterions leaching out of cross-linked fibers. 

Peptide Synthesis for Materials Manufacturing 

This invention focuses on peptides for making materials. Characterizing peptide-based materials and determining relationships between polymer structure and solution and material properties matter most here. The scalability of polymer synthesis and materials manufacture; convenience; cost of synthesis and purification; and limitations on poly› mer structure are important. 

Linear homopolypeptides and heteropolypeptides, uni› form and non-uniform sequence, respectively, can be pre› pared by solid-phase or solution-phase methods (chemical approaches) or recombinant methods (biological approaches) (see FIG. 3). These synthesis approaches have advantages and disadvantages for different purposes. For example, chemical synthesis is advantageous for small› peptide biologics production. Solution-phase approaches are usually favored for therapeutic peptides shorter than 15 amino acid residues and quantities over 100 kg, whereas complex or longer sequences are usually made by solid› phase synthesis, and peptides longer than 50 residues are made by recombinant methods (Thayer, 2011 ). All industrial enzymes are made by biological methods. 

Ring-opening polymerization of a-amino acid N-car› boxyanhydrides, the main approach to synthesizing peptides in solution, is utilized herein. The method yields statistical, linear polypeptides. Growth occurs only by the probabilistic addition of monomers to active chain ends. Monomers are present throughout the process, but their concentration falls with time. Polymer mass and yield are functions of mecha› nism and other reaction particulars. The product will nor› mally have a Poisson distribution of chain lengths, (Ne/N) Nexp(-N), where N is degree of polymerization (DP) and N is number-average DP (Strobl, 2007). N can range from 2 to >2,000. Short-range order is possible; long-range order, improbable. Synthesis from a heterogeneous monomer population is expected to yield chains of virtually identical amino acid composition. The approach is advantageous if monodisperse polymers and sequence specificity are optional. Polydisperse co-poly(L-glutamic acid4, L-tyrosine 1) (PLEY), a random copolymer, has been studied (Khadka et al., 2011; Haynie et al., 2012; Haynie et al., 2013). 

Solution-phase production of a monodisperse population of polypeptides of identical sequence is not only possible but actually done in industry. It is very expensive, however, if the DP is greater than about 15 residues. Solid-phase syn› thesis of sequence-specific polypeptides is even more expen› sive per unit mass. DP is practically limited to about 50 residues, as yield decreases with chain length. Double coupling can improve the efficiency of monomer addition at each step and the yield of desired product, albeit at a cost. Consequently, peptides made by chemical methods cannot be competitive in the marketplace unless consumers are willing to pay a premium. In general terms, sequence specificity by chemical production is too expensive except for limited purposes in medicine, basic research and defense (see Bray, 2003). 

The functional properties of proteins are "microscopic" in that they typically depend on amino acid sequence. For instance, enzyme activity requires appropriate positioning of chemical groups. The physical properties of polymer aggre› gates, by contrast, are "macroscopic." Such properties could depend on amino acid sequence as much as composition. 

Perhaps the most positive way of regarding solution-phase synthesis of peptides in a materials context is to focus on cases in which it is certain that sequence will be less relevant than a) amino acid composition, b) predictability of water solubility, c) measurability of number-average mass and weight-average molecular mass and therefore polydispersity index and d) reproducibility of polymer production and materials processing. Chemical composition, average DP and, to a lesser extent, polydispersity index may be treated as essentially continuous and independent variables, and the systematic investigation of relationships between polymer structure and materials will be possible. 

Chemical synthesis, it should be acknowledged, presents both advantages and disadvantages. The plus side features ease of polymer preparation and latitude with regard to composition. A potential advantage for materials is the incorporation of unusual amino acids, which proceeds in essentially the same way as for a usual amino acid. The main limitations are the prohibitive cost of controlling sequence. For this subject invention, however, specific sequences are not needed. Polydispersity can be controlled by reaction conditions and fractionation. Side chain protecting groups can influence polymerization, but the final amino acid com› position of products can be measured. Incomplete removal of protecting groups after synthesis can influence down› stream processing, but the abundance of such groups can be quantified. How material properties depend on a lack of sequence uniformity or polydispersity can be tested by recombinant production of monodisperse peptides of iden- tical composition. 

Recombinant production will in any case have unique and complementary advantages to solution-phase synthesis for materials research and possible technology commercializa› tion. If control over amino acid composition, sequence and polydispersity are required at any scale of production, if the ability to encode manufacturing instructions into polymer structure requires sequence specificity, and if there is no need to guarantee freedom from small amounts of bacterial contaminant, then recombinant production could be the least expensive approach to synthesis. Peptide purity of >95% is readily and reliably achieved, and polymers of substantially greater purity can be obtained without great difficulty. Sys› tematic variation of amino acid composition is more difficult than for solution-phase synthesis, as at least one gene is needed per polymer structure, but it can be done by estab› lished and cost-effective methods. A small number of rep› resentative random sequences of a given amino acid com› position and chain length could be made by recombinant methods and compared experimentally. A blend of different recombinant random sequences can prove useful for mate› rial properties. Biological peptide production is also poten› tially significant for sustainable manufacturing. Production in photosynthetic bacteria can be especially advantageous for the purpose; high yields of recombinant proteins have been obtained in photosynthetic bacteria. 

One approach to peptide synthesis and processing could be more advantageous for some amino acid compositions than others. Recombinant synthesis may be best for protein› like sequences, that is, ones for which the composition resembles the average composition of proteins. Whether a solution-phase approach or a recombinant approach for random peptide production is best for large-scale production depends on the cost of polymer production, characterization and processing, and the desirability of the physical, chemical or biological properties displayed by materials that can actually be made with a polypeptide of a given sequence or composition. 

