The COVID-19 pandemic has killed millions of people worldwide since its outbreak in
December 2019. The pandemic is caused by the SARS-CoV-2 virus whose main protease (Mpro)
is a promising drug target since it plays a key role in viral proliferation and
replication. Currently, developing an effective therapy is an urgent task, which
requires accurately estimating the ligand-binding free energy to SARS-CoV-2 Mpro.
However, it should be noted that the accuracy of a free energy method probably depends
on the protein target. A highly accurate approach for some targets may fail to produce a
reasonable correlation with the experiment when a novel enzyme is considered as a drug
target. Therefore, in this context, the ligand-binding affinity to SARS-CoV-2 Mpro was
calculated via various approaches. The molecular docking approach was manipulated using
Autodock Vina (Vina) and Autodock4 (AD4) protocols to preliminarily investigate the
ligand-binding affinity and pose to SARS-CoV-2 Mpro. The binding free energy was then
refined using the fast pulling of ligand (FPL), linear interaction energy (LIE),
molecular mechanics-Poisson–Boltzmann surface area (MM-PBSA), and free energy
perturbation (FEP) methods. The benchmark results indicated that for docking
calculations, Vina is more accurate than AD4, and for free energy methods, FEP is the
most accurate method, followed by LIE, FPL, and MM-PBSA (FEP > LIE ≈ FPL >
MM-PBSA). Moreover, atomistic simulations revealed that the van der Waals interaction is
the dominant factor. The residues
Thr26
,
His41
,
Ser46
,
Asn142
,
Gly143
,
Cys145
,
His164
,
Glu166
, and
Gln189
are essential elements affecting the binding process. Our
benchmark provides guidelines for further investigations using computational
approaches.
The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.
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