An improved strategy for efficient expression and purification of soluble HIV-1 tat protein in <Emphasis Type="Italic">E.coli</Emphasis> |
| |
Authors: | Shi-meng Zhang Rong Fan Tian-yi Yang Yi Sun Jing-yun Li Qin-zhi Xu Ping-kun Zhou |
| |
Institution: | 1. The Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China 2. Institute of Microbiology and Epidemiology, Beijing 100071, China |
| |
Abstract: | Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity
has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat
protein in E.coli, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare E.coli codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare E.coli codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six
rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged
Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein
were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the
transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification
of soluble Tat protein has paved the way to fully investigate its exogenous function. |
| |
Keywords: | HIV tat gene E coli Protein expression Codon usage |
本文献已被 维普 万方数据 SpringerLink 等数据库收录! |
|