IMMUNE PROPERTIES IN THE PRODUCTED RECOMBINANT SIP YOUNG 6 HIS PROTEIN FROM THE SEA STAR IGKAPPA GENE THROUGH HEK 293 EBNA CELLS REVEALING AGAINST SPIKE-RBD PROTEIN AND HRP ONE CHANGES IN SEA STAR GENE EXPRESSION?

In 2014 we have isolated and cloned the sea star Igkappa gene which induced an anti-HRP primitive invertebrate antibody. We attempt, in the present work, to produce a SIP Young 6 His protein through HeK 293 EBNA cells after Igkappa gene cloning. This protein bound HRP by the use of Escherichia coli as vector of cloning but not HRP with HeK cells as vector. Is there changes in sea star IGKappa gene expression? We notice also that the SARS-CoV-2-SPIKE RBD Protein didn’t bind to SIP Young 6 His protein when compared to HRP.


INTRODUCTION
The sea star IGKappa gene was discovered in Osteraset et al. (2014).It is an IPA (Invertebrate Primitive Antibody) which produce an anti-HRP protein.We recall it was obtained from sea star Asterias rubens immunized to HRP (Horse-radish peroxydase).In Leclerc and Otten (2014) it was inserted in an Escherichia coli plasmid and showed a specific binding to the antigen HRP (Leclerc & Otten, 2014).Recently, (Leclerc, 2021) we have also inserted the sea star IGKappa gene in Hek 293 EBNA cells as it was done by Kujirai et al. (2014).In the present work we attempt to observe if these last cells produce the anti-HRP protein immobilzed in magnetic beads by contact to HRP.A control which is made of another protein: the recombinant SARS-CoV-2-Spike .RBD was used to verify the binding of this last one to anti-HRP protein issued from HeK 293 cells.A comparison is performed between the binding of anti-HRP protein to HRP and the binding of anti-HRP to spike RBD protein.

Subcloning in Expression Vector
The genes coding for the target proteins were chemically synthesized with optimization for expression in HEK293 cells.The sequence is illustrated below (Osteraset et al., 2014).Assay was performed as a co-immunoprecipitation by using the sea star anti-HRP protein instead a classical antibody.The binding of sea star protein (Bait protein) and target proteins or Prey proteins (HRP, Recombinant Sars-Cov-2 Spike RBD Flag Tag (R and D Products) were assessed by Western-blots.Flag Tag was the following amino-acid terminaison: DYKDDDDK.

Sequences Information
Experiments were realized by the use of magnetic beads in column.These last ones were bound to bait protein.Secondly the bait protein linked or not to prey one.A same molar amount of proteins was used for bait and prey proteins sometimes a ratio: 1/2 was performed At this point only the sea star protein and the protein bound to it remain immobilized to the magnetic beads.A specific elution with buffer containing Imizadole was performed and determined the specificity of the binding when compared to negative controls.

Small-scale Expression and Purification Tests Short Protein Purification Protocol Description
Culture medium was purified by affinity against His-tag (IMAC) by a standard method: -Equilibration with PBS, pH 7.5 -Wash 1/2/3 with PBS, pH 7.5, 0 mM, 30mM and 50mM imidazole buffer -Elution with PBS, pH 7.5, 200mM and 400mM imidazole buffer -Analysis by SDS-PAGE of fractions of interest -Final sample QC: qualitative by SDS-PAGE, quantitative by Bradford method E2-E6 were pooled, buffer exchanged vs PBS, pH7.5 by dialysis method.The purification test results and QC are illustrated in Figure 1 and Figure 2.

SIP Young -6 HIS Does Not Bind Spike RBD Protein
Immunoprecipitation assay doesn't reveal a binding between SIP Young 6 HIS protein and Spike RBD one.Eluted fractions don't contain a labelling at all in Western-blots.

SIP Young-6 HIS and HRP Protein
Two assays were performed: A pull-down (PD) and an outside (O) ones.Unfortunately they show no specific binding between the 2 proteins in a ratio 1/1 and in the ratio 1/2 (2 HRP for 1 SIP Young-6 HIS) in Eluates: EluO and Elu PD.
All the fractions have been loaded in a Western-Blot.The excess of proteins is named FT1 in Pull-Down assay, FT2 in the case of Outside assay.W1 corresponds to washing 1 with buffer and W5 to washing 5 Results are summarized as following in Figure 3. -FT2 and W5 fractions for the pull-down assay There is no signal on the elution lanes for both assays.We note, on the other hand that SIP-Young -6HIS protein doesn't bind SARS-CoV -2-Spike RBD protein: this result is more logic in our mind.

Figure 2 .
Figure 2. The final QC gel.Coomassie blue staining.A. Reduced PAGE analysis.B. Western blot with anti-his antibody (ECL revelation).MW.Molecular weight marker.These figures show clearly that the eluates E2,E3, E4 contain mainly the SIP Young 6 His protein which is revelated by the anti-His antibody (ECL revelation)