Protein purification is a crucial step in experiments studying protein performance and structure. E. coli lysis, as the first step in purification, aims to efficiently release proteins from the cell into the supernatant while preserving protein activity to the greatest extent possible. Currently, mainstream E. coli lysis methods are divided into physical and chemical methods, each with its own advantages and disadvantages, but also certain limitations.
Physical methods include sonication, homogenization, grinding, and freeze-thaw cycles. Their advantages include not introducing exogenous impurities, lower cost, and high lysis efficiency, but the operation process is relatively cumbersome, and mechanical force can easily damage the target protein structure. Chemical methods encompass organic solvent methods, acid-base lysis methods, and lysozyme methods, and are not limited by sample volume, processing samples from microliters to liters. However, some chemical reagents may affect protein activity, and some methods have poor reproducibility.
Addressing the pain points of traditional methods, a novel E. coli lysis solution has emerged, achieving the need for rapid, mild, and low-cost lysis, providing a superior solution for protein purification pretreatment. This lysis solution is a single-component formulation, simple to operate, and has good reproducibility, requiring no complex equipment. Its core components possess highly efficient lysis capabilities: lysozyme can penetrate the bacterial cell wall and degrade it into cell wall disaccharides within 1 minute, while nuclease can rapidly digest bacterial DNA and RNA within 1 minute, degrading them into oligonucleotide fragments. This dual action significantly enhances lysis efficiency.
The significant advantages of this lysis buffer are that it effectively reduces the viscosity of the supernatant after lysis, avoiding difficulties in subsequent processing due to nucleic acid residue; the mild lysis conditions inhibit the degradation of the target protein, maximizing the preservation of protein activity and integrity, while improving sample clarification, laying a good foundation for subsequent column purification, and reducing the risk of endotoxin contamination.
Experiments have verified its superior performance: tests using green fluorescent protein as a sample showed that the lysis effect gradually improved with increasing lysis buffer addition ratio or prolonged lysis time. A 1× concentration is the recommended dosage, and the effect can be further improved by adjusting the dosage or time. Comparative experiments were conducted on two different proteins, treated with ultrasound and this novel lysozyme lysis method, respectively. Electrophoresis results showed that the target protein obtained by the novel lysis method had fewer impurities below the band, and the purity after elution was significantly higher than that obtained by ultrasound.
Compared to traditional ultrasonic lysis methods, the core advantages of the novel lysate can be summarized in five points: First, it is easy to operate, with a single-component formulation that eliminates the need for complex ratios and is easy to learn; second, it is time-saving and efficient, with lysis completed in as little as ten minutes after addition, reducing the time by more than half compared to conventional methods; third, it produces superior protein activity and purity, with gentle conditions avoiding mechanical damage to proteins and ensuring experimental quality; fourth, it optimizes the purification process, with nucleases degrading nucleic acids to improve clarity and accelerate column loading; and fifth, it has broad compatibility, being EDTA-free and suitable for purification using non-tolerant metal chelate chromatography media, making it applicable to a wider range of scenarios.
