Expression and purification of AtRALF1 from Escherichia coli


  • Benjamin T Lanham Shepherd University
  • Jonathan Gilkerson Shepherd University



protein expression, peptides, Rapid Alkalinization Factor


Rapid Alakalinization Factors (RALFs) are a family of plant specific peptide growth factors involved in myriad plant processes including growth, development, and response to stresses. These secreted peptides are genetically encoded and processed into an active form from a larger pre-pro-peptide. The active forms are ~5 kDa and have two disulfide bridges.  Here we report our attempts to express the active form of AtRALF1 (At1g02900) from the soluble fractions of two different expression strains of E. coli, Origami 2 (DE3) and SHuffleT7 pLysY.  Both of these strains are commercially available and genetically altered for expression of proteins with disulfide bonds. Expression of these peptides is challenging because misfolded disulfides or other structural issues result in insoluble peptides. We sought to optimize expression conditions to bulk purify HIS(6x)-AtRALF1 by cobalt column chromatography. Under most conditions the peptide was insoluble.  However, at 25oC around 50% of total HIS(6x)-AtRALF1 was soluble in Origami cells. As controls, we expressed mutated versions with alterations in the conserved YISY motif which are physiologically inactive.  Surprisingly, these mutant versions were 100% soluble in most conditions tested.  All versions of HIS(6x)-AtRALF1 were observed to migrate ~12 kDa in SDS-PAGE gels although their predicted molecular weights is ~8 kDa. Treatment under strong reducing conditions had no effect on this migration pattern.  We conclude that AtRALF1’s insolubility issues could be more related to a structure in the YISY motif than to the formation of disulfide bonds.

Author Biographies

Benjamin T Lanham, Shepherd University

Department of Chemistry

Jonathan Gilkerson, Shepherd University

Assistant Professor

Department of Biology




How to Cite

Lanham, B. T., & Gilkerson, J. (2019). Expression and purification of AtRALF1 from Escherichia coli. Proceedings of the West Virginia Academy of Science, 91(1).



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