Modified GDF9 Monomer for Granulosa Cell Proliferation
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Solution Overview
Problem
Current methods for producing cumulin, a heterodimer of growth and differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15), face challenges such as reduced protein expression levels, co-purification of homodimers, and variable potency due to difficulties in optimizing the ratios of GDF9 and BMP15, which affects its use in assisted reproductive technologies like in vitro maturation (IVM).
Innovation Solution
Development of a cumulin-like GDF9 protein with specific amino acid substitutions, such as S363R, K366G, and N369H, that enhance binding to activin-like kinase 4 and 5 receptors, allowing for the production of a homogenous preparation that consistently activates Smad-2/3 signaling pathways, thereby promoting granulosa cell proliferation and differentiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional cumulin (GDF9:BMP15 heterodimer) is produced by co-expression of both proteins, then biological activity is enhanced, but production complexity increases and homogeneity decreases due to co-purification of homodimers
Solution Approach 1:
The invention extracts and transfers the critical functional elements (amino acid residues S363, K366, N369) from BMP15 to the GDF9 protein sequence, creating a modified GDF9 that can function alone without requiring BMP15 co-expression. This eliminates the complexity of producing and purifying heterodimers while maintaining biological activity.
Solution Approach 2:
The modified GDF9 protein acquires multiple functions: it retains the native GDF9 activity while also gaining the ability to activate Smad-2/3 signaling pathways typically associated with BMP15. This single protein performs what previously required a complex heterodimeric system.
2Reliability
If ratios of GDF9 and BMP15 are optimized for heterodimer formation, then potency increases, but manufacturing precision requirements increase
Solution Approach 1:
The invention changes the amino acid sequence parameters of GDF9 by introducing specific substitutions (S363R, K366G, N369H) that alter its functional properties. These parameter changes enable the protein to achieve high potency without requiring precise control of protein ratio parameters during manufacturing.
3Reliability
If modified GDF9 with amino acid substitutions is produced, then binding affinity to ALK4/5 receptors and Smad-2/3 activation increases, but protein structure complexity increases
Solution Approach 1:
The invention applies local quality changes by introducing amino acid substitutions at specific positions (S363, K366, N369) within the GDF9 protein sequence. These localized modifications enhance receptor binding affinity and signaling activity without requiring global changes to the overall protein structure or production complexity.
Data Source
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AI summary
An agent capable of promoting proliferation and differentiation of granulosa cells is disclosed which comprises a growth and differentiation factor-9 (GDF9) protein comprising a modified GDF9 polypeptide monomer which includes at least one amino acid substitution that enhances binding to and/or activation of activin-like kinase 4 and/or 5 receptor (ALK4/5). The agent is preferably provided in a mature dimeric form (eg comprising two monomers of the same modified GDF9 polypeptide monomer) and/or in a pro/mature complex form. The agent may be suitable for, inter alia, promoting oocyte maturation in vitro for use in assisted reproductive technologies.