Tatiana Ilina1*, Ana Pantelić1, Dejana Milić1, and Marija Vidović1
1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia
tanyailina134 [at] gmail.com
Abstract
Ramonda serbica Panč., an ancient resurrection plant, can withstand extended periods of desiccation and reestablish metabolic activity shortly upon watering. One of the key players of desiccation resistance in resurrection species is the accumulation of protective late embryogenesis abundant proteins (LEAPs). During cellular dehydration, these intrinsically disordered proteins (IDPs) may stabilize the native structures of proteins and membranes. Differential transcriptome and proteome analyses revealed that members of the LEA4 protein family represent the majority of desiccation-inducible LEAPs.
The aim of this work was to in silico characterize and recombinantly produce a member of the LEA4 protein family group – LEA_301. This protein is predicted to be highly disordered (above 85%). Its sequence is composed of predominantly charged and polar amino acids (39% of the sequence is charged, 12% of the sequence is lysine), with acidic pI (5.92). According to secondary structure predictors (JPred, PsiPred, Phyer2, Sopma, and FELLS), LEAP_301 exhibits high propensity to form amphipathic α-helix (above 95 %). This can be important for their function during desiccation, when this protein may undergo a disorder-to-order transition, and stabilize biomolecules. To experimentally validate the secondary structure of this protein and assume its physiological role, we produced high yield and purity of LEA_301 by recombinant DNA technology.
Bacterial cells (Escherichia coli BL21(DE3)) were transformed with the vector with the LEA_301-6xHis gene, and optimization of protein expression was done. Final purification was done by immobilized metal ion affinity chromatography. The sequence was validated by Western blot and mass spectrometry. Obtained LEA_301 will be structurally characterized using methods for secondary structure determination such as Fourier-transform infrared spectroscopy and circular dichroism spectroscopy and compared with the in silico results.
Keywords: bioinformatics, LEA proteins, secondary structure prediction, intrinsically disordered proteins, production of recombinant proteins
Acknowledgement: This work was funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Contract No. 451-03-66/2024-03/200042) and by the Science Fund of the Republic of Serbia-RS (PROMIS project LEAPSyn-SCI, grant no. 6039663).