Scaffolding of cystine-stabilized miniproteins

S. Sankaran, Ivan Stojanovic, A. Barendregt, A.J.R. Heck, Richardus B.M. Schasfoort, Pascal Jonkheijm

Research output: Contribution to journalArticleAcademicpeer-review

2 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)1039-1046
Number of pages8
JournalChemistrySelect
Volume1
Issue number5
DOIs
Publication statusPublished - 2016

Keywords

  • IR-103908
  • METIS-320704

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