Sandwiched-fusion Strategy Facilitates Recombinant Production Of Small Labile Proteins

Efficient production of large quantities of soluble,properly folded proteins is of high demand in modern structural and functional genomics.Despite much advancement toward improving recombinant protein expression,many eukaryotic proteins especially small peptides with disorder structure often fail to be recovered due to rapid proteolytic degradation.As a result,related structural and functional studies has been greatly hindered by the absence of a robust method allowing efficient production of labile proteins.Here we show that the sandwiched-fusion strategy,which is based on two protein tags incorporated both at the amino-and carboxyl-terminus of target protein,could be employed to overcome this obstacle.We have exploited this strategy on heterologous expression in Escherichia coli of nine small degradation-prone eukaryotic proteins.including eight mitochondria-derived peptides(MDPs)from human:humanin,six humanin-like peptides(SHLP1-6)and the MOTS-C,and a degradation-prone transcription factor Guy1.MDPs behave as novel signal peptides in cells and play a diverse role in many biological processes including apoptosis,cell survival,substrate metabolism,inflammatory response,and response to stressors.Since the discovery of the first MDP nearly two decades ago,to date,however,all MDP samples used for research were chemically synthesized;and the structural and functional studies have been greatly hampered by the high cost.We show here that the sandwiched-fusion strategy,which provides robust protection against proteolysis,affords an economical method to obtain large quantities of pure six MDPs,in sharp contrast to otherwise unsuccessful recovery using the traditional amino-fusion method.Further biophysical characterization and interaction studies by NMR spectroscopy confirmed that the proteins produced by this novel approach were properly folded into their biologically active structures.The versatility of this novel strategy was further demonstrated by successful preparation of the sex-determining mosquito transcription factor,Guyl,another labile peptide whose recombinant production has yet to be achieved.We therefore anticipate this strategy could be widely utilized in production of other labile protein systems.