In recent years, various strategies have been used to overexpress and purify HIV-1 protease because it is an essential drug target in anti-retroviral therapy. Obtaining sufficient quantities of the enzyme, however, remains challenging. Overexpression of large quantities is prevented due to the enzyme’s autolytic nature and its inherent cytotoxicity in Escherichia coli cells. Here, we describe a novel HIV-1 protease purification method using a thioredoxin–hexahistidine fusion system for the wild-type and two variant proteases. The fusion proteases were overexpressed in E. coli and recovered by immobilised metal ion affinity chromatography. The proteases were cleaved from the fusion constructs using thrombin. When compared to the standard overexpression and purification protocol in use in our laboratory, the expression of the fusion-derived wild-type protease was increased from 0.83 to 2.5 mg/l of culture medium. The expression levels of the two variant proteases ranged from 1.5 to 2 mg/l of culture medium. The fusion wild-type and variant proteases were inactive before the cleavage of the thioredoxin–hexahistidine fusion tag as no enzymatic activity was observed. The proteases were, however, active after cleavage of the tag. The novel thioredoxin–hexahistidine fusion system, therefore, enables the successful overexpression and purification of catalytically active HIV-1 proteases.
The Protein Journal – Springer Journals
Published: Jun 4, 2018
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera