Batroxobin Purification via pH-Conductivity Chromatography Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The production of batroxobin from snake venom is hindered by high cost, low yield, and low enzyme activity due to protease degradation in yeast host systems, limiting its widespread application.
Innovation Solution
An expression and purification method involving pH and conductivity adjustment, cation and anion chromatography, unidirectional tangential-flow filtration, and gel filtration chromatography to obtain high-purity active batroxobin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If batroxobin is extracted from snake venom, then the enzyme can be obtained, but the production cost is too high and the yield is low
Solution Approach 1:
The patent uses genetic engineering to copy the batroxobin gene and express it in yeast cells (Pichia pastoris), creating a biological factory that produces batroxobin through fermentation rather than extraction from snake venom. This copying approach enables large-scale production with lower costs and higher yields compared to traditional venom extraction methods.
2Ease of manufacture
If batroxobin is expressed in E. coli, then the production cost is reduced, but the expression level is very small and most forms are inclusion bodies
Solution Approach 1:
The patent uses yeast (Pichia pastoris) as an intermediary expression system between E. coli and mammalian cells. Yeast provides eukaryotic post-translational modification capabilities including correct disulfide bond pairing and glycosylation, while maintaining ease of cultivation and lower production costs compared to mammalian cell systems. This intermediary system achieves both high expression levels and proper protein folding.
3Manufacturing precision
If batroxobin is expressed in eukaryotic systems, then correct disulfide bond pairing is ensured, but the process complexity increases
Solution Approach 1:
The patent uses Pichia pastoris, a simple eukaryotic yeast system, as a disposable expression platform that provides necessary post-translational modifications without the complexity of mammalian cell systems. The yeast can be easily cultivated, harvested, and discarded after use, providing correct disulfide bond pairing and glycosylation at lower operational complexity.
4Productivity
If yeast fermentation is used to produce batroxobin, then the yield is improved, but the enzyme activity decreases due to protease degradation
Solution Approach 1:
The patent applies preliminary anti-action by adding protease inhibitors (PMSF and leupeptin) to the fermentation system before protease degradation can occur. These inhibitors prevent endogenous yeast proteases from degrading the expressed batroxobin, thereby maintaining high enzyme activity alongside high yield. The inhibitors are added at the beginning of the expression process to preemptively block the harmful proteolytic activity.
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
The method enhances enzyme activity and yield, achieving a coagulation effect similar to commercially available batroxobin, with improved purity and reduced process time.
Implementation Method 1
Taking the supernatant of the yeast fermentation broth of batroxobin, and adjusting pH value and conductivity; Performing chromatography over a cation resin
Implementation Method 2
Performing chromatography over an anion resin
Implementation Method 3
Performing unidirectional tangential-flow filtration and concentration
Implementation Method 4
Performing gel filtration chromatography to concentrated protein solution
Data Source
Figure 1~2
Figure 3~4
Figure 5
AI summary
Provided is an expression and purification method for recombinant batroxobin. By means of adjustment and optimization on the pH value and conductivity of a culture medium supernatant, a target protein can well bind to a chromatography filler, whereas most of impurity proteins cannot bind to the filler and thus flow through same, thus improving the purity of a collected liquid; therefore, the method can suppress the activity of a hydrolase in a fermentation supernatant, and further ensures no flow-through of the target protein, thereby ensuring a high yield. Further, integrating cation chromatography, anion chromatography, unidirectional tangential-flow filtration and concentration and gel filtration chromatography for purification obtains high-purity active batroxobin protein. A simple salt-gradient elution mode is replaced by a salt-gradient and pH-gradient composite elution mode, thus greatly improving the protein purity. The purification method uses a continuous-flow purification process, and an intermediate product does not need to undergo pH value and conductivity regulation, dilution, liquid change and other operations, thereby achieving advantages of shortened process time, improved enzyme activity, reduced storage tanks, workers and plant area, etc. In-vitro and in-vivo verification shows that the batroxobin purified by the method of the present invention has blood coagulation effect similar to that of commercially available natural batroxobin, thereby achieving extensive application prospects.