Modified MIS Protein Cleavage Design for Higher Recombinant Yield
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Solution Overview
Problem
The production of recombinant human Mullerian Inhibiting Substance (MIS) protein is inefficient, leading to low yield and incomplete cleavage, which affects its bioactivity and therapeutic potential, particularly in treating cancers and conditions characterized by excess androgen.
Innovation Solution
Engineered recombinant human MIS protein with a modified Kex cleavage site and a non-MIS leader sequence, such as a human serum albumin leader sequence, to enhance cleavage efficiency and yield, optionally incorporating a FLAG tag for purification and labeling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type human MIS protein is produced using standard recombinant methods, then the protein can be obtained, but the cleavage efficiency is incomplete and the yield is low
Solution Approach 1:
The patent modifies the amino acid sequence at the cleavage site (changing basic residues to acidic residues) to alter the biochemical parameters of the protein, thereby improving both cleavage efficiency and yield without requiring complex additional processing steps
Solution Approach 2:
The patent removes the problematic basic residues (arginine or lysine) at the cleavage site that interfere with proper processing, extracting the harmful element that prevents efficient cleavage and yields
2Reliability
If the normal MIS leader sequence is used, then the protein follows natural processing, but cleavage efficiency remains suboptimal
Solution Approach 1:
The patent changes the biochemical parameters of the leader sequence by substituting amino acids to create optimal recognition sites for proteolytic cleavage, ensuring both complete processing and high production yields
3Ease of manufacture
If standard purification methods are used without tags, then the process is simpler, but purification efficiency and labeling capability are reduced
Solution Approach 1:
The patent introduces a FLAG tag as an intermediary element that facilitates purification through immunaffinity methods and enables labeling capabilities, while the tag is designed to be removable if needed, thus providing ease of manufacture when required
Data Source
AI summary
The present invention relates to modified recombinant human MIS protein which has improved cleavage and increased bioactivity and increased potency as compared to wild-type human MIS protein. Other aspects of the invention relate to methods to prevent and treat cancers, such as cancers that express the MIS receptor type II (MISRII) by administering to a subject a composition comprising a recombinant human MIS protein. Another aspect of the present invention relates to methods to lower plasma androgen levels in a subject, and/or for the treatment of a subject with a disease characterized by excess androgen. Another aspect provides pharmaceutical compositions and kits and methods for use comprising a recombinant human MIS protein. Another aspect of the present invention relates to methods to decrease the dose of a chemotherapeutic agent by administering the chemotherapeutic agent with the recombinant MIS protein that lowers the effective dose of the chemotherapeutic agent.