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Bioactive site-specifically modified proteins for 4D patterning of gel biomaterials


Protein-modified biomaterials can be used to modulate cellular function in three dimensions. However, as the dynamic heterogeneous control over complex cell physiology continues to be sought, strategies that permit a reversible and user-defined tethering of fragile proteins to materials remain in great need. Here we introduce a modular and robust semisynthetic approach to reversibly pattern cell-laden hydrogels with site-specifically modified proteins. Exploiting a versatile sortase-mediated transpeptidation, we generate a diverse library of homogeneous, singly functionalized proteins with bioorthogonal reactive handles for biomaterial modification. We demonstrate the photoreversible immobilization of fluorescent proteins, enzymes and growth factors to gels with excellent spatiotemporal resolution while retaining native protein bioactivity. Localized epidermal growth factor presentation enables dynamic regulation over proliferation, intracellular mitogen-activated protein kinase signalling and subcellularly resolved receptor endocytosis. Our method broadly permits the modification and patterning of a wide range of proteins, which provides newfound avenues to probe and direct advanced cellular fates in four dimensions.

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Fig. 1: Generation of sortagged protein library for biomaterial modification.
Fig. 2: Comparing the activity of differently modified proteins.
Fig. 3: Photopatterned alteration of hydrogel biomaterials with sortagged proteins.
Fig. 4: 4D photoevolution of hydrogel biomaterials patterned with multiple sortagged proteins.
Fig. 5: Spatial patterning of gels with bioactive site-specifically modified enzymes and growth factors.
Fig. 6: Modulating cell fate with a photoreleasable sortagged fluorophore growth factor chimeric protein.

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Data availability

Plasmids and data that support the findings of this study are available from the corresponding author upon reasonable request.


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The authors recognize and thank R. Seifert and D. Hailey of the University of Washington Garvey Imaging Center for their ongoing support and advice, F. Watt and T. Hiratsuka (King’s College London) for helpful discussion on visualizing the MAPK activation, M. Matsuda (Kyoto University) for a gift of the HeLa cells transfected with the EKAREV FRET sensor, as well as R. Warden-Rothman and A. Tsourkis (University of Pennsylvania) for providing the pSTEPL plasmid37. The authors thank B. Badeau for assistance in synthesizing N3-oNB-OSu, S. Adelmund for providing BCN-OSu and A. Im for help with the protein purification optimization. We acknowledge support from S. Edgar at the UW Mass Spectrometry Center as well as that from the NIH and N. Peters at the UW W. M. Keck Microscopy Center (S10 OD016240). This work was supported by a University of Washington Faculty Startup Grant (C.A.D.), a Jaconette L. Tietze Young Scientist Research Award (C.A.D.) and a CAREER Award (DMR 1652141, C.A.D.) from the National Science Foundation.

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Authors and Affiliations



For this manuscript, J.A.S. and C.A.D. conceived and designed the experiments; J.A.S. and G.M.B. performed the experiments; J.A.S. and C.A.D. analysed the data and prepared the figures; J.A.S. and C.A.D. wrote the paper.

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Correspondence to Cole A. DeForest.

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Supplementary Information

Supplementary Figs. 1–23, Supplementary Table 1, Supplementary Methods, Supplementary Video Captions 1–3, Supplementary References 1–5.

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Supplementary Video 1

Scanned xy planes of multiphoton ‘cell’ pattern from Fig. 4f–i rendered using Imaris.

Supplementary Video 2

Scanned xz planes of multiphoton ‘cell’ pattern from Figure 4f–i rendered using Imaris.

Supplementary Video 3

Time-lapse FRET quantification of HeLa cells expressing EKAREV.

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Shadish, J.A., Benuska, G.M. & DeForest, C.A. Bioactive site-specifically modified proteins for 4D patterning of gel biomaterials. Nat. Mater. 18, 1005–1014 (2019).

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