Cost‐effective purification process development for chimeric hepatitis B core (HBc) virus‐like particles assisted by molecular dynamic simulation
Authors: Jingxiu Bi
Shuang Yin
Yingli Wang
Zhiguo Su
Bingyang Zhang
Journal: Engineering in Life Sciences (2021)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: hepatitis B core; molecular dynamic simulation; protein characterization; protein purification; virus‐like particle.
Affiliations: School of Chemical Engineering &amp; Advanced Materials, Faculty of Engineering, Computer and Mathematical Sciences University of Adelaide  Adelaide SA Australia, School of Chinese Medicine and Food Engineering Shanxi University of Traditional Chinese Medicine  Jinzhong Shanxi Province P. R. China, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering Chinese Academy of Sciences  Beijing P. R. China

Abstract:  Inserting foreign epitopes to hepatitis B core (HBc) virus-like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost-effective purification process was developed for two chimeric HBc VLPs displaying Epstein-Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core-HBc was found to be less stable in water environment compared with EBNA1-HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of α-helix of HCV core-HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost-effective purification approach. 

Retracted: No

PubPeer comments:
 Figure 1B. * Red boxes: the top parts of lanes 5 and 6 look more similar to each other than expected. * The position of the thick bands around 27 kDa in lanes 5-7 is at an awkward angle with respect to the rest of the lanes Could the authors please share a high resolution photo of the original gel? ![file] , 
 Figure 1C and Figure 3B appear to be the same photo. The legend of Figure 1C only describes 13 lanes. Could the authors please check if this should have shown a different gel? ![file] , 
 Figure 3. * Blue boxes: The top parts of lane 5 in Figure 3A and lane 11 in Figure 3B look very similar, but the 27 kDa band has a different thickness. Can the authors explain how these two lanes, each being shown as part of a different gel, can look so similar, please? ![file] , 
 #2 Sorry for the mistake. When doing the combination of the pictures a wrong SDS-PAGE gel picture was put there in Figure 1C. Attached is the correct SDS-PAGE gel for Figure 1C with 13 lanes. We will inform the editor to fix the figure as soon as possible.![file] , 
 It is a careless mistake that lane 5 in Figure 3A is not correct. The figure was made by combine lanes form different original gel pictures after change the color to black and white. The original pictures were put together in different layers and when moving the lane 5 in Figure 3A, wrong layer was selected and only partial of the lane was selected and moved. Sorry that we didn t carefully check every lane in the figure. Now after checking, we found that actually the correct lane for lane 5 in Figure 3A should be the lane 2 in Figure 3B and the correct lane 2 was not shown. In addition, the Mw marker lane in Figure 3B is not correct either. We have now fixed the figure as attached. Original SDS-PAGE gel pictures for lane 5-7 in Figure 3A, lane 2-4 and lane 11-13 in Figure 3B are attached. We will inform the editor to fix the figure as soon as possible.![file] ![file] ![file] , 
 #1 The left lane in the red box showed the total protein in the bacteria lysate and right lane is the supernatant of the bacteria lysate after centrifugation. Similar impure protein bands are expected. The original picture is attached and after cropping and change color to black and white, it can make these two lines more similar. About the angle, please see attached original picture.![file] , 
 #1 The left lane in the red box showed the total protein in the bacteria lysate and right lane is the supernatant of the bacteria lysate after centrifugation. Similar impure protein bands are expected. The original picture is attached and after cropping and change color to black and white, it can make these two lines more similar. About the angle, please see attached original picture. ![file] , 
 Dear Dr. Zhang, thank you for posting original photos of the gels. Could you please post them again and clarify which lanes in the originals correspond to the published figures? I do not see any red boxes in comment #7, and the gels shown in #6 and 7 do not appear to correspond to the published figures, except for one or two lanes. Also, the explanation of the partially shifted layers is not satisfactory. According to the Instructions for authors, undisclosed lane splicing is not allowed. See: https: onlinelibrary.wiley.com pb-assets assets 16182863 ELS_Instructions_to_authors_2020-1624347140367.pdf  Cropping of gels, or re-positioning of lanes fields, is permitted providing that all alterations are clearly indicated by the use of dividing lines in the image itself, vital data are not removed, an explanation of the alterations is included in the figure legend and images of full blots or gels that the figures are derived from are supplied in the supporting information. Unacceptable manipulation includes, but is not limited to, the enhancement of one feature band over others, removal of background noise bands, etc. It seems that the published figures appear to be a composite of many different gels, which makes it hard to compare band sizes and exposures. ,