Modified Signal Peptide for Inclusion Body Protein Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The challenge is to efficiently express and purify proMMP-7 in E. coli without proteolytic degradation, as existing methods result in low expression levels and yield due to the protein's toxicity and susceptibility to proteases, making it difficult to produce this protein for medical applications.
Innovation Solution
A nucleic acid fragment with a modified signal peptide, such as the PhoA-alkaline phosphatase signal peptide with specific amino acid substitutions at positions 13 and 21, is used to inhibit proteolytic degradation and enhance expression levels of proMMP-7, allowing for its efficient production as an inclusion body in E. coli.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a signal sequence is attached to the N-terminal of proMMP-7 to secret it into periplasm, then the protein can be transported across the inner membrane, but the expression level remains low and the protein is susceptible to proteolytic degradation
Solution Approach 1:
The patent modifies specific amino acid residues in the signal sequence (Ala21 to Asp/Glu/Lys/His/Phe/Tyr, and Leu13 to Pro/Phe/Trp) to change the parameters of signal peptidase recognition and cleavage efficiency. These parameter changes in the signal sequence structure enable high-level expression while preventing proteolytic degradation, resolving the contradiction between transport capability and expression efficiency.
2Ease of operation
If a signal sequence is attached to secret proMMP-7 into periplasm, then the protein can be transported, but it becomes susceptible to proteolytic degradation by periplasmic proteases
Solution Approach 1:
The modified signal sequence takes preliminary anti-action by preventing proteolytic degradation before it can occur. The specific amino acid substitutions (Ala21 to charged/aromatic residues and Leu13 to Pro/Phe/Trp) create a signal sequence that is resistant to signal peptidase cleavage, thereby preventing proteolytic degradation in advance and ensuring protein stability in the periplasm.
3Productivity
If antibiotics are used for selection of recombinant cells, then the production efficiency can be maintained, but the process becomes toxic and poses safety concerns
Solution Approach 1:
The patent extracts and eliminates the harmful element (antibiotics) from the production process. By using a modified signal sequence that ensures high-level expression and stability of proMMP-7, the invention makes antibiotic selection unnecessary, thereby removing the toxic component while maintaining production efficiency through improved protein expression and stability.
Data Source
Figure 1~2
Figure 3~4
Figure 5
AI summary
A process for preparing an inclusion body-forming protein is provided. A nucleic acid fragment consisting of a nucleotide sequence coding for a modified alkaline phosphatase signal peptide (modified APSP) where leucine at the 13th position in the amino acid sequence shown in SEQ ID NO: 1 is substituted with proline and/or alanine at the 21st position is substituted with the other amino acid, downstream of which nucleotide sequence is bound a nucleotide sequence of a gene of a protein of interest, and a process for preparing an inclusion body-forming protein consisting of the following steps: (1) preparing an expression vector in which a nucleic acid fragment is incorporated consisting of a nucleotide sequence coding for a modified signal peptide downstream of which is bound a nucleotide sequence of a gene of a protein of interest, (2) preparing a host cell transformed with the expression vector that produces an inclusion body-forming protein, and (3) purifying the inclusion body-forming protein from culture obtained by culturing the host cell that produces the inclusion body-forming protein. The above other amino acid is selected from the group consisting of aspartic acid, glutamic acid, lysine, histidine, phenylalanine and tyrosine.