Bacillus subtilis bFGF Production via Trans-Splicing Intein
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Solution Overview
Problem
Current methods for expressing exogenous polypeptides, such as bFGF, in Bacillus subtilis face challenges including low yield, instability, and high production costs due to endotoxin contamination and protease secretion, which hinder their commercial application in pharmaceuticals.
Innovation Solution
A nucleic acid construct comprising a trans-splicing intein from Anabaena DNA polymerase III unit and a short peptide affinity tag is used to express bFGF in Bacillus subtilis, allowing for efficient and cost-effective production by incorporating a 6x His tag and utilizing sequential cation exchange and heparin-agarose chromatography for purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If E. coli is used as a bacterial host to express recombinant proteins, then the production cost is low and growth rate is fast, but the purified recombinant protein is accompanied by a large amount of endotoxin which leads to toxic effects
Solution Approach 1:
The invention extracts and removes the harmful endotoxin component from the production system by switching from E. coli (gram-negative with LPS outer membrane) to Bacillus subtilis (gram-positive without endotoxins). This extraction of the harmful element allows production to continue at low cost while eliminating the toxic contamination problem.
Solution Approach 2:
The invention uses Bacillus subtilis as a disposable host system that naturally lacks endotoxins. By choosing a different bacterial host that is inherently free from the harmful substance, the need for expensive endotoxin removal kits is eliminated, maintaining cost-effectiveness while solving the contamination issue.
2Object-affected harmful factors
If Bacillus subtilis is used to express exogenous proteins, then endotoxin contamination is avoided, but proteases are secreted which adversely affect the stable expression and yield of exogenous proteins
Solution Approach 1:
The invention applies preliminary action by using an inducible promoter system (tac promoter with IPTG induction) to control protease expression. The protease is activated only after the exogenous protein has been synthesized and accumulated, preventing proteolytic degradation during the critical expression phase while still allowing for downstream processing.
Solution Approach 2:
The invention implements periodic action through time-separated expression phases: first the exogenous protein is expressed under inducible control, then protease activity is activated in a subsequent phase. This periodic separation of functions allows high-yield protein accumulation followed by controlled proteolysis for release or processing.
3Ease of operation
If exogenous proteins are secreted into the medium by Bacillus subtilis, then the host bacteria growth is affected, but this also affects the high-efficiency expression of exogenous proteins
Solution Approach 1:
The invention uses preliminary action by first synthesizing and accumulating the exogenous protein intracellularly under inducible control, then subsequently activating protease activity to facilitate controlled release. This prevents the growth inhibition problem by separating protein synthesis from secretion timing.
Solution Approach 2:
The invention extracts the secretion function from the expression phase by using a two-step process: intracellular synthesis followed by controlled proteolytic release. This separation allows high-efficiency expression without the negative effects of concurrent secretion on bacterial growth.
4Reliability
If routine replacement of fresh medium containing commercially available bFGF is used in stem cell culture, then the therapeutic effect is maintained, but the R&D costs are greatly increased
Solution Approach 1:
The invention enables self-service by engineering the stem cell culture system to produce its own required growth factor (bFGF) through the integrated Bacillus subtilis expression system. The bacteria continuously supply the necessary protein, eliminating the need for external medium replacement and reducing dependency on commercial products.
Solution Approach 2:
The invention applies universality by creating a multi-functional system where Bacillus subtilis serves both as a production platform and an in-situ source of therapeutic protein. The same bacterial system that produces the protein can be directly integrated into the culture medium, providing both production and delivery functions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach results in high-yield, biologically active bFGF production with reduced costs and minimal endotoxin contamination, suitable for large-scale cultivation and commercial use.
Implementation Method 1
Inteins are protein elements that are capable of self-cleavage from host proteins and catalyzed flanking sequences (extein) linked by peptide bonds. The self-cleavage process is called 'protein splicing'.
Implementation Method 2
sequential cation exchange and heparin-agarose chromatography for purification
Implementation Method 3
sequential cation exchange and heparin-agarose chromatography for purification
Data Source
AI summary
The present invention relates to the preparation of human basic fibroblast growth factor by using Bacillus subtilis and endonuclease. Specifically, the present invention provides a nucleic acid construct, which comprises an insert, and the insert comprises, from the 5′ end to the 3′ end, a polynucleotide sequence that encodes a short peptide affinity tag, a trans-splicing intein derived from Anabaena and an exogenous polypeptide; and wherein the short peptide affinity tag serves as an N-terminal extein of the trans-splicing intein, and the exogenous polypeptide serves as a C-terminal extein of the trans-splicing intein. The present invention further provides an expression vector and a host cell that comprise the construct, and a method for producing and purifying foreign proteins. The expression system and method of the present invention can significantly improve the expression efficiency of biologically active exogenous proteins, reduce the generation of inclusion bodies, simplify purification steps, greatly reduce purification costs, and are especially suitable for large-scale cultivation.