Scaffolding of Cystine‐Stabilized Miniproteins

Abstract

Biomolecular scaffolds were engineered by genetically fusing robust miniproteins in a sequence, like a chain. By fusing these miniprotein chains to a teal fluorescent protein (TFP), an efficient strategy was devised for their production in E. coli. Miniproteins that bind β‐trypsin, VEGF and HIV‐1 Nef proteins were used to make 4 individual chains, each with 3 miniproteins arranged in different orders. Their stability and binding properties were analyzed with native ESI‐mass spectrometry, microscale thermophoresis, surface plasmon resonance imaging and a trypsin activity assay. Miniproteins within the chains were found to be functional and significantly robust. The trypsin activity assay showed that miniprotein chains containing two β‐trypsin inhibitors clearly inhibit β‐trypsin more than two fold stronger compared to chains containing only one β‐trypsin inhibitor indicating that multivalent interactions occurs. SPR imaging results indicated that these constructs had the potential to simultaneously bind multiple target proteins. However, each miniprotein chain exhibited different binding affinities towards the target proteins, especially VEGF and HIV‐1 Nef possibly due to issues dealing with folding, miniprotein orientations, non‐specific adhesion and slow proteolytic degradation of the miniproteins. Analysis of these issues provided insights into parameters that need to be taken into consideration while designing such multi‐specific protein constructs. By careful optimization, such constructs have the potential to be used in a very versatile manner for various molecular engineering applications.

Thermophoresis for characterizing biomolecular interaction

“…MST and fluorescence correlation spectroscopy (FCS) techniques were used to measure binding constant where the dissociation constants that determined by MST and F K d = 236 nM and K d = 282 nM, respectively. Different applications [43][44][45][46][47][48][49][50] of MST technique in the characterization of protein-protein interactions are summarized in Table 1.…”

Cell Adhesion on RGD-Displaying Knottins with Varying Numbers of Tryptophan Amino Acids to Tune the Affinity for Assembly on Cucurbit[8]uril Surfaces

“…These knottin constructs were genetically fused to the C-terminus of a teal fluorescent protein (TFP), separated by an enterokinase cleavage site (EKCS) linker and expressed in E. coli to prevent inclusion in body formation as described previously. , Once expressed, the knottins were cleaved from TFP using enterokinase and purified using centrifugal filters. (See the Supporting Information for details.)…”