Rao Xian C, Li Shu, Hu Jin C, Jin Xiao L, Hu Xiao M, Huang Jian J, Chen Zi J, Zhu Jun M, Hu Fu Q
Department of Microbiology, College of Medicine, The Third Military Medical University/Key Lab of Microbial Engineering Under the Educational Committee in Chongqing, 400038, PR China.
Protein Expr Purif. 2004 Jul;36(1):11-8. doi: 10.1016/j.pep.2004.01.020.
Peptide antibiotics are often hard to express in engineered bacteria at high level. According to the properties of peptide antibiotics, a heterologous protein PaP3.30, encoded by ORF30 of Pseudomonas aeruginosa bacteriophage PaP3, was selected as a carrier molecule. The gene of the carrier molecule was constructed into the plasmid pQE-32 to give rise to the vector pQE-PaP30 for expression of peptide antibiotics in Escherichia coli. A his-tagged fusion protein was genetically constructed with a peptide antibiotic at its carboxy terminus. The novel carrier molecule was used for high-level expression of six peptide antibiotics with different sizes and isoelectric points in E. coli, which are hPAB-beta, MSI-78, Melletin, hBD-1, Cecropin A, and an ovine anion peptide. And further, one of six peptide antibiotics, hPAB-beta (an analog of a human peptide antibiotic), was taken as an example for studies of recovery of interesting products from the fusion partner, purification and antimicrobial activity evaluation. The results indicated that the expressed fusion protein existed as an inclusion body in the cytoplasm and the expression amounts of six peptide antibiotic fusions are all higher than 34% of the total cell protein. The expression products could be easily purified by Ni-NTA chromatography. Cyanogen bromide was used to cut at the methionine linker between the carrier and hPAB-beta peptide. hPAB-beta was recovered from the fusion partner and purified to homogeneity with High S cation-exchange and Bio-gel P6 gel chromatography. The bactericidal activities of the purified recombinant hPAB-beta against P. aeruginosa are 31-64 microg/ml, and against Staphylococcus aureus are > or = 128 microg/ml, being comparable to that of the chemical synthesis peptide. These results show that the carrier molecule can result in high-level expression of peptide antibiotics, and expression products can be easily recovered from their fusion partner and retain their bioactivity.
肽类抗生素通常难以在工程菌中高水平表达。根据肽类抗生素的特性，选择了铜绿假单胞菌噬菌体PaP3的ORF30编码的异源蛋白PaP3.30作为载体分子。将载体分子的基因构建到质粒pQE-32中，得到载体pQE-PaP30，用于在大肠杆菌中表达肽类抗生素。在羧基末端将带His标签的融合蛋白与一种肽类抗生素进行基因构建。该新型载体分子用于在大肠杆菌中高水平表达六种不同大小和等电点的肽类抗生素，分别是hPAB-β、MSI-78、蜂毒素、hBD-1、天蚕素A和一种绵羊阴离子肽。此外，以六种肽类抗生素之一的hPAB-β（一种人肽类抗生素类似物）为例，研究了从融合伙伴中回收目标产物、纯化及抗菌活性评估。结果表明，表达的融合蛋白以包涵体形式存在于细胞质中，六种肽类抗生素融合蛋白的表达量均高于总细胞蛋白的34%。表达产物可通过Ni-NTA层析轻松纯化。使用溴化氰在载体与hPAB-β肽之间的甲硫氨酸连接部位进行切割。通过High S阳离子交换和Bio-gel P6凝胶层析从融合伙伴中回收hPAB-β并纯化至均一。纯化后的重组hPAB-β对铜绿假单胞菌的杀菌活性为31 - 64μg/ml，对金黄色葡萄球菌的杀菌活性≥128μg/ml，与化学合成肽相当。这些结果表明，该载体分子可实现肽类抗生素的高水平表达，表达产物可从其融合伙伴中轻松回收并保留其生物活性。
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