Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag.
The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggregating peptide ELK16. In this scheme, the target proteins were fused in the C-termini with cSAT and expressed as insoluble aggregates. After intein self-cleavage, target proteins were released into the soluble fraction with high yield and reasonable purity. We demonstrated the applicability of this scheme by preparing seven model viral peptides, with lengths ranging from 32 aa to 72 aa. By adding an N-terminal thioredoxin tag, we enhanced the yield of target peptides released from the aggregates. The purified viral peptides demonstrated high antigenic activities in ELISA and were successfully applied to dissecting the antigenic regions of influenza hemagglutinin. The cSAT scheme described here allows for the rapid and low-cost preparation of multiple antigenic peptides for immunological screening of a broad range of viral antigens.
Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing 100084, China.
Wanghui
W
Xu
Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing 100084, China.
Qing
Q
Zhao
Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing 100084, China.
Lei
L
Xing
Department of Chemical Engineering, Tsinghua University, One Tsinghua Garden Road, Beijing 100084, China.
Zhanglin
Z
Lin
D000956:Antigens, Viral / Q000096:biosynthesis*; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis*; D008967:Molecular Biology / Q000379:methods*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D000956:Antigens, Viral; D015699:HIV Envelope Protein gp120; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D010455:Peptides; D011993:Recombinant Fusion Proteins