Solubilisation and purification of recombinant bluetongue virus VP7 expressed in a bacterial system

Bluetongue virus viral protein 7 stability in the presence of glycerol and sodium chloride

“…Intrinsic tryptophan fluorescence emission spectra collected confirmed that BTV VP7 has a native like tertiary structure even in the presence of 8 M urea ( Fig. 2A ), with BTV VP7 in 2 M urea, 8 M urea, and 5 M urea with 300 mM sodium chloride all showing a similar tertiary structure, with a peak at approximately 341 nm, which is consistent with BTV VP7 in 0 M and 5 M urea (without the presence of any of the additives evaluated in this study) seen in Russell and Gildenhuys [ 19 ]. Increased fluorescence is seen at 295 nm for BTV VP7 in 2 M urea, when compared to 8 M urea and 5 M urea with 300 mM sodium chloride, with the lowest fluorescence at 295 nm seen in 6 M guanidinium chloride ( Fig.…”

“…BTV VP7 was expressed, solubilized and purified using the methods defined in NiCo21 (DE3) Escherichia coli cells [ 19 ]. The protein samples were visualized and quantified using a reducing SDS-PAGE (polyacrylamide gel electrophoresis) with 15% separating gel and 4% stacking gel.…”

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA)

“…In some cases, the recombinant protein was not even purified [26,27,40] and the yield of recombinant protein obtained at the end of the production process was not always mentioned, as well as the method of protein quantification [23][24][25]. Some authors reported the yield obtained, that was characterized, from one author to the other, by an extreme variability [9,20,40,44,45]. Since we preferred to optimize the recVP7 production from supernatant of infected Sf9, because of its easy, rapid and costless production, we can not compare the yield of our recVP7 with the other ones previously published, these last being mostly related to recombinant proteins isolated from cell lysates.…”

“…VP7, a ~ 40 kDa protein arranged as trimers to form the BTV core surface [5][6][7][8], is conserved within all the different BTV serotypes and is used for serodiagnostic purpose to detect infected animals [15,16]. A variety of recombinant DNA methods have been used to express BTV VP7 protein [9,[17][18][19][20][21] and among these the baculovirus expression system has been successfully used to reach a high yield production of the biologically active recombinant VP7 protein [9,18]. This method is preferred because it is able to overcome the protein solubility problems that usually occur when prokaryotic expression systems are used [19,20,22].…”

“…A variety of recombinant DNA methods have been used to express BTV VP7 protein [9,[17][18][19][20][21] and among these the baculovirus expression system has been successfully used to reach a high yield production of the biologically active recombinant VP7 protein [9,18]. This method is preferred because it is able to overcome the protein solubility problems that usually occur when prokaryotic expression systems are used [19,20,22]. In this work, we describe a recombinant BTV VP7 production method using a baculovirus expression system and a subsequent purification method of the protein by immobilized metal affinity chromatography (IMAC).…”