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目的分离纯化嗜酸乳杆菌(Lactobacillus acidophilus)ATCC 4356的3-磷酸甘油醛脱氢酶。方法利用硫酸铵分级沉淀法、亲和层析、阴离子交换和分子筛层析对嗜酸乳杆菌GAPDH进行了分离纯化。通过N端测序鉴定了纯化产物;并用分子筛测定了嗜酸乳杆菌GAPDH的相对分子质量(Mr)。结果纯化产物在SDS-PAGE胶上呈现Mr为3.7×104的单一条带,产量为0.9 mg/L(嗜酸乳杆菌培养物)。N端测序结果证明纯化产物为嗜酸乳杆菌GAPDH。分子筛测定的GAPDH的Mr为7.08×104,表明嗜酸乳杆菌GAPDH是二聚体。结论通过该纯化方案,可大量获得电泳纯的嗜酸乳杆菌GAPDH,为进一步研究其免疫功能以及开发为免疫促进剂打下了基础。
Objective To isolate and purify glyceraldehyde 3 - phosphate dehydrogenase from Lactobacillus acidophilus ATCC 4356. Methods The Lactobacillus acidophilus GAPDH was isolated and purified by ammonium sulfate fractionation, affinity chromatography, anion exchange and molecular sieve chromatography. The purified product was identified by N-terminal sequencing. The relative molecular mass (Mr) of Lactobacillus acidophilus GAPDH was determined by molecular sieve. Results The purified product showed a single band with Mr of 3.7 × 104 on SDS-PAGE gel with a yield of 0.9 mg / L (Lactobacillus acidophilus culture). N-terminal sequencing proved that the purified product was Lactobacillus acidophilus GAPDH. Mr of GAPDH determined by molecular sieve was 7.08 × 104, indicating that Lactobacillus acidophilus GAPDH is a dimer. Conclusion The purified Lactobacillus acidophilus GAPDH can be obtained in large quantities, which lays the foundation for further research on its immune function and development as an immunostimulant.