Control of the spatial arrangement of proteins on surfaces is essential in a number of emerging technologies, including protein microarrays, biosensors, 1 tissue engineering, and regenerative medicine. 2 Patterning is also a powerful tool in cell biology, wherein cell arrays are used to elucidate the factors that mediate migration, proliferation, and cell-cell interactions. 3 Although photolithography holds a preeminent place as a patterning method in the microelectronics industry, optical lithography of proteins has been hampered by the need either to use traditional chemical photoresists or to modify proteins chemically by attachment of photoreactive functional groups; both methods can compromise protein function. 4Production of a protein "photoresist" without the need for post-translational chemical modification would require an intrinsically photoreactive protein. Recently, the incorporation of photo-reactive, non-canonical amino acids into proteins via site-specific 5 and residuespecific techniques has been reported. 6 Here we describe the microbial expression of artificial proteins bearing the photosensitive non-canonical amino acid para-azidophenylalanine (pN 3 Phe). The recombinant proteins, designated artificial extracellular matrix proteins with aryl azides (aECM-N 3 ), belong to a family of engineered proteins designed to exhibit mechanical properties similar to those of native elastins 7 and to support adhesion of mammalian cells through cell-binding domains (CS5 or RGD) derived from fibronectin ( Fig 1A). 8 These proteins can be crosslinked efficiently upon irradiation at 365 nm. The physical properties of the crosslinked films can be controlled by changing the pN 3 Phe content, and thin films can be patterned on surfaces via photolithographic techniques. We demonstrate the utility of the method by creating cell arrays through selective cell attachment to photolithographically prepared protein patterns. aECM-N 3 variants were expressed in Escherichia coli cultures supplemented with pN 3 Phe (Supporting Information). Incorporation of pN 3 Phe into the recombinant proteins relies on activation of the photosensitive amino acid by the phenylalanyl-tRNA synthetase (PheRS) of the bacterial expression host. The PheRS used for this study was a previously characterized mutant with relaxed substrate specificity. 9 This method results in statistical decoding of phenylalanine (Phe) codons placed at regular intervals in the coding sequence. 9 Proteins were expressed in a Phe-auxotrophic E. coli strain and purified by exploiting the temperaturedependent phase behavior of proteins that contain elastin-like repeats. 10 Incorporation efficiency was determined by integration of the aromatic proton signals in the 1 H NMR spectra of the purified proteins; the extent of Phe replacement varied from 13% to 53%, depending on the concentration of pN 3 Phe in the expression medium (Supporting Information). Understanding the response of the photoreactive protein to irradiation is crucial for highresolution pattern formation....

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Thin films of controlled elastic modulus were made by photocrosslinking artificial extracellular matrix (aECM) proteins containing the photosensitive amino acid para-azidophenylalanine (pN 3 Phe). The elastic moduli of the films were calculated from nanoindentation data collected by atomic force microscopy (AFM) using a thin-film Hertz model. The modulus was shown to be tunable in the range 0.3-1.0 MPa either by controlling the irradiation time or by varying the level of pN 3 Phe in the protein. Tensile measurements on bulk films of the same proteins and finite-element simulation of the indentation process agreed with the thin-film modulus measurements from AFM. Substrates characterized by spatial variation in elastic modulus were created by local control of the irradiation time.