A Novel Method For Chemical Synthesis Of Proteins Enabled By Classical Peptide Motif CCPGCC And Its Application

Compared with the traditional methods of chemical total synthesis and introducing unnatural amino acids（u AAs）by codon expansion technology,the chemical protein semi-synthesis owns the flexibility of introducing u AAs including special post-translational modification by solid-phase peptide synthesis,as well as the facile manipulation and high efficiency of recombinant expression.Chemical protein semi-synthesis enabled by fragment condensation—especially most widely used native chemical ligation（NCL）—provides a powerful technical platform for obtaining the proteins with various functions.One of the substrates of NCL,recombinant protein thioester or the chemically more stable acyl hydrazides,can be obtained in vitro through intein fusion technology or enzymatic reactions,but these methods are extremely dependent on the correct folded structure of the recombinantly expressed protein.In fact,most of the protein fragments are expressed in Escherichia coli as unfolded or misfolded inclusion bodies,which is difficult to guarantee the refolding effect in vitro or water solubility.As such,there is an increasing interest in developing chemical approaches to obtain recombinant proteinα-thioesters or hydrazides,which could be performed regardless of protein’s folding state.One of these available methods,S-cyanylation-mediated hydrazinolysis is theoretically not limited by the C-terminal hydrazinolysis site of protein of interest（POI）.The key issue here is,however,that cysteine S-cyanylation is non-selective,allowing no other cysteine residue in the POI’s sequence.To achieve regioselective S-cyanylation-mediated hydrazinolysis,we developed an orthogonal and reversible thiol protection system with the aid of the biarsenical compound Fl As H-EDT₂ targeted modification to the CCPGCC tag.Therefore,we termed this strategy as tetracysteine enabled protein ligation（TCEPL）.In this strategy,the CCPGCC sequence and purification tags（like hexahistidine tag）are fused to the C-terminus of POI.After the CCPGCC tag is specifically protected by Fl As H,another temporal protection for the remaining cysteines would be adopted using certain reagents,like phenacyl group（Pac）selected here.Next,the addition of excess ethanedithiol（EDT）would be allowed to strip the Fl As H off the target proteins within minutes,which enables the subsequent regioselective S-cyanylation-mediated hydrazinolysis.Finally,the Pac-tagged hydrazide participates in NCL,followed by the removal of Pac to obtain the target（semi）synthetic protein.The TCEPL strategy developed in this work has the following innovative points and advantages:1)highly regioselective S-cyanylation;2)not limited by the folding state of the recombinant proteins;3)not limited by the reaction site for traceless ligation or C-terminal labeling;4)larger application scope of S-cyanylation-mediated hydrazinolysis for complementing the chemical protein synthesis toolbox.In this paper,the feasibility of the TCEPL strategy in three one-pot operations including thiol orthogonal protection,S-cyanylation-mediated hydrazinolysis and NCL–de Pac was first verified with model peptides synthesized by SPPS.Encouraged by the above results,we then tested the TCEPL strategy with the fusion proteins expressed in E.coli as inclusion bodies,and successfully completed the efficient semi-synthesis of an iron-sulfur protein—pf Rd and a 207-aa matrix metalloproteinase 14（MMP-14）under denaturing conditions,demonstrating the viability and generality of our strategy.Besides,our strategy could be extended to the labeling of POIs like ubiquitin-like protein 5（UBL5）with amino reagents bearing a variety of functional groups,indicating that TCEPL is highly versatile for traceless labeling at POI’s C-terminus.In addition,we took the thrombin exosite I inhibitor,142-aa triabin,as the（semi）synthetic target,and comprehensively compared the differences on producing triabin（1–109）-NHNH₂containing 4 cysteine residues between the traditional intein-mediated hydrazinolysis and our TCEPL strategy.We found that the hydrazide fragments produced by hydrazinolysis of intein would co-precipitate with the unreacted substrate,resulting in low efficiency and purification yield,while Pac-tagged protein hydrazide could be obtained smoothly through TCEPL strategy under denaturing condition.However,the separation of the target hydrazide fragments by HPLC and subsequent purification of NCL remain quite challenging due to the properties of triabin including its aggregation tendency.Nevertheless,these works have provided valuable technique support for the semi-synthesis of sulfotyrosine-containing triabin in the future.Finally,in order to explore the interaction mechanism of natural thrombin inhibitors—triabin,we optimized its E.coli expression and purification methods without the high-cost thrombin-affinity purification.The disulfide linkages were confirmed through digestion and LC-ESI-MS analysis,and the recombinant triabin is as competent as hirudin variant 1（HIRV1）in inhibiting thrombin’s fibrinogenolytic activity,showing the reliability of our recombinantly expressed protein.Most interestingly,the structure-function studies through extensive site-directed mutagenesis reveal that triabin’s Phe-106 involved in the hydrophobic contacts plays a surprisingly important role in the thrombin inhibition,in contrast to the negatively charged residues Asp-135 or Glu-128 involved in the salt-bridge interaction.As such,the current work complements our understanding on the interaction mechanism of natural thrombin inhibitors that might be of interest to develop more reliable antithrombin peptide therapeutics.