Optimizing HIV-1 protease production in Escherichia coli as fusion protein

Abstract

BackgroundHuman immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations.ResultsA recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis) and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1Pr per liter.ConclusionsBy using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.

A single mutation in the core domain of the lac repressor reduces leakiness

“…A tethered dimer of HIV protease [46], which cannot be produced in soluble form under WT LacI control due to its toxicity [47,48], was fused to maltose-binding protein (MBP) and cloned in place of GFP to give plasmids pLIHP and pLIHP_W220F. In the absence of the inducer, cells transformed with the pLIHP_W220F plasmid showed growth curves that are indistinguishable from those of cells expressing GFP (Figure  7, panel A).…”

Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

“…Currently, lac operon regulation is a research objective in systems biology, and lac operon elements are used as tools in biotechnology and synthetic biology [4-7]. For this reason, a detailed knowledge about the lac operon induction behavior is required.…”

Codon Optimisation Is Key for Pernisine Expression in Escherichia coli

“…The final yields of the lyophilised pernisine co and pernisine S355Aco were around 10 mg per litre of culture. Indeed, the use of such codon-optimised genes is becoming more attractive, and recently more examples of improved overexpression of such proteins have been reported [ 25 , 26 , 27 ]. Although there have been further studies carried out, to date, improvements to heterologous protein expression using codon-optimisation or by supplying extra tRNAs remain more or less empirical [ 28 ].…”