Method for the immobilization of biologically active polypeptides by using maltose binding protein
Inventors
Kim, Sang-Heon • Kim, Soo Hyun • Han, Min
Assignees
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Publication Number
US-8669075-B2
Publication Date
2014-03-11
Expiration Date
Abstract
The present invention relates to a method for immobilization of a biologically active polypeptide using maltose binding protein (MBP) and a biologically active solid substrate on which a biologically active polypeptide is immobilized by the above method. More particularly, the present invention relates to a method for immobilization of a biologically active polypeptide comprising the following steps; 1) preparing a fusion protein by linking a biologically active polypeptide to carboxyl terminal of maltose binding protein (MBP); and 2) immobilizing the fusion protein on the hydrophobic surface by physical adsorption of amino terminal containing hydrophobic domain exposed on the surface of maltose binding protein on the hydrophobic surface of a solid substrate, and a biologically active solid substrate on which a biologically active polypeptide is immobilized by the said method.
Core Innovation
The patent describes an immobilization method for a biologically active polypeptide in which the polypeptide is fused to maltose-binding protein (MBP) and immobilized on a hydrophobic surface of a solid substrate by physical adsorption. In the VEGF example, VEGF is linked to the carboxyl terminal of MBP, and an amino terminal hydrophobic domain of MBP is exposed to provide hydrophobic contact with the hydrophobic surface.
The method includes preparing a fusion protein from a nucleic acid sequence encoding the fusion of VEGF and MBP, where the VEGF is amplified and then ligated to a nucleic acid sequence encoding MBP. The patent states that the VEGF portion immobilized on the solid substrate retains biological activity, including quantitative retention thresholds.
The patent further describes immobilization of MBP-fused VEGF on hydrophobic substrates, including hydrophobic polystyrene surfaces, and characterizes adsorption behavior using QCM. It also states that immobilized VEGF can induce cell morphology changes such as pseudopodia and related shape changes in response to VEGF stimulation.
Claims Coverage
The independent claim covers a method for immobilizing VEGF on a solid substrate by constructing an MBP fusion that places VEGF at the carboxyl terminal of MBP and immobilizing the fusion on a hydrophobic surface via physical adsorption of an MBP amino-terminal hydrophobic domain. The claim set adds constraints related to immobilization conditions, quantitative biological activity retention, VEGF target specificity, purification, and example hydrophobic surface materials.
VEGF–MBP fusion with VEGF linked to the carboxyl terminal of MBP
The method ligates an amplified nucleic acid sequence encoding VEGF to a nucleic acid sequence encoding maltose binding protein (MBP) to make a fusion protein of VEGF and MBP, wherein the fusion protein is a VEGF linked to a carboxyl terminal of MBP.
Immobilizing via physical adsorption of the MBP amino-terminal hydrophobic domain on a hydrophobic solid substrate
The fusion protein is immobilized on the hydrophobic surface by physical adsorption of an amino terminal containing hydrophobic domain exposed on the surface of MBP on the hydrophobic surface of a solid substrate.
Preparing the fusion protein from the nucleic acid sequence encoding the fusion protein
The method includes preparing the fusion protein from the nucleic acid sequence encoding the fusion protein.
Amplifying VEGF nucleic acid using PCR primers SEQ ID NO: 1 and SEQ ID NO:2
The method includes performing a PCR reaction with PCR primers of SEQ ID NO: 1 and SEQ ID NO:2 to amplify a nucleic acid sequence encoding VEGF.
The inventive core is the MBP fusion architecture that links VEGF to the carboxyl terminal of MBP and uses the MBP amino-terminal hydrophobic domain for physical adsorption-based immobilization on a hydrophobic solid substrate.
Stated Advantages
Retention of biological activity of the immobilized VEGF portion on the solid substrate, including thresholds such as at least 50%.
Documented Applications
Regenerative medicine, stem cell differentiation and tissue engineering, cell chips/censors, and biosensors/high-throughput screening.
Cell culture applications in which immobilized VEGF induces phosphorylation-related signaling and cell morphology changes, including pseudopodia and cell shape changes.
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