Cloning and Expression of H. influenzae 49247 IgA Protease in E. coli.

Abstract

IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H. influenzae 49247 IgA protease showed unique DNA and amino acid sequence but with typical endopeptidase domain and beta transporter domain compared with known IgA proteases from the same species. To facilitate expression and purification, the H. influenzae 49247 IgA protease gene was sub-cloned into the pET28-A(+) vector with insertion of a 6xHis tag downstream of the endopeptidase domain and upstream of the potential autocleavage site. The recombined IgA protease can be constitutively expressed in E. coli and secreted into the culture medium. With a simple nickel affinity binding, the secreted IgA protease can be purified with high purity (95%) and a molecular weight of about 130 kDa. The identity of the IgA protease was validated by the presence of 6xHis tag in the purified protein by western blotting and its ability to cleave human IgA1 molecule. Collectively, the successful cloning, expression and purification of H. influenzae 49247 IgA protease will augment its therapeutic study in IgAN treatment.

• Authors: Honglian Wang, Xia Zhong, Jianchun Li, Menglian Zhu, Lu Wang, Xingli Ji, Junming Fan, Li Wang
• Journal: Molecular biotechnology (Mol Biotechnol) Vol. 60 Issue 2 Pg. 134-140 (Feb 2018) ISSN: 1559-0305 [Electronic] United States
• PMID: 29318516 (Publication Type: Journal Article)

Chemical References

• DNA Primers
• His-His-His-His-His-His
• Immunoglobulin A
• Oligopeptides
• Recombinant Fusion Proteins
• Histidine
• Serine Endopeptidases
• IgA-specific serine endopeptidase

Topics

• Amino Acid Sequence
• Chromatography, Affinity
• Cloning, Molecular (methods)
• DNA Primers (chemical synthesis, genetics)
• Escherichia coli (enzymology, genetics)
• Gene Expression
• Genetic Vectors (chemistry, metabolism)
• Glomerulonephritis, IGA (therapy)
• Haemophilus influenzae (enzymology, genetics)
• Histidine (genetics, metabolism)
• Humans
• Immunoglobulin A (chemistry, metabolism)
• Molecular Weight
• Oligopeptides (genetics, metabolism)
• Protein Domains
• Proteolysis
• Recombinant Fusion Proteins (chemistry, genetics, metabolism)
• Sequence Alignment
• Sequence Homology, Amino Acid
• Serine Endopeptidases (chemistry, genetics, metabolism)