Heterologous expression and purification of recombinant protein TROL for the purpose of antiserum production in rabbits

Regulatorni gen At4g01050 smješten na 4. kromosomu biljke Arabidopsis thaliana (L.) Heynh kodira za protein TROL (thylakoid rhodanase-like protein). TROL je integralni membranski protein potreban za vezanje FNR-a (feredoksin-NADP+ reduktaze) i održavanje visoke efikasnosti linearnog toka elektrona u fotosintezi. Heterologna ekspresija dopušta proizvodnju biljnih proteina u organizmu koji je različit od prirodnog izvora. Za povećanu ekspresiju proteina TROL odabran je ekspresijski vektor pRSET A, u kojem je uklonirani gen pod kontrolom T7 promotora. Soj E. coli BL21(DE3)pMico transformiran je ekspresijskim vektorom pRSET A/miniTROL te je ekspresija proteina inducirana dodatkom IPTG-a. Dobiveni rekombinantni protein je zatim pročišćen afinitetnom kromatografijom u nativnim uvjetima i korišten kao antigen za proizvodnju antiseruma u zečevima. Ovako dobiveni antiserum obično je specifičniji i imunoreaktivniji nego antitijela razvijena na kratke sintetske peptideThe regulatory gene At4g01050 is located on the 4th chromosome of Arabidopsis thaliana (L.) Heynh, encoding for protein TROL (thylakoid rhodanase-like protein). TROL is an integral membrane protein required for tethering of FNR (ferredoxin-NADP+ reductase) and sustaining efficient linear electron flow in vascular plants. Heterologous expression allows the production of plant proteins in an organism which is simpler than the natural source. For the increased expression of TROL protein, pRSET A vector has been selected, into which our gene of interest is cloned under the control of the T7 promotor. E. coli strain BL21(DE3)pMico was transformed with the pRSET A/miniTROL construct and expression was induced by the addition of IPTG. The resulting recombinant protein was then purified by affinity column chromatography under native conditions and has been used as an antigene for antiserum production in rabbits. The resulting antiserum is expected to be more specific and immunoreactive than the antibodies raised against shorter synthetic peptides