Facile Expression And Purification Of Therapeutic Protein(Peptide)Using The Cleavable Self-Aggregating Tag Scheme

Protein（peptide）drug is one of the major class of biopharmaceutical with more than 100listed drugs.The global market size was valued at approximately$100 billion and growth rapidly.However,there are serious bottlenecks in the preparation of many protein（peptide）drugs,e.g.expression protein drug in E.coli often encounters the problem of expression as inactive inclusion bodies,and chemical method is inefficient and costly if larger than 30 amino acids.Based on the protein（peptide）expression and purification technology cleavable self-aggregating tag（cSAT）that developed by our previous work,this study focusses on the production of one protein drug human growth hormone（hGH）and two peptide drugs insulin-like growth factor 1（IGF-1）and Brain natriuretic peptide（BNP）.cSAT scheme is composed of a self-assembling short peptide and a self-cleaving intein.Herein,for the efficient preparation of hGH,four self-assembling short peptides:a surfactant-like peptide L₆KD,coiled coil peptideα3-peptide and aβ-sheet short peptide EFK8,ELK16 were test in the cSAT scheme.All the four self-assembling peptides could drive the hGH fusion proteins into active aggregates when expression in E.coli BL21（DE3）and obtain hGH to be active after in vitro cleavage.The optimal conditions for hGH fusion of L₆KD,α3-peptide and EFK8 were 0.2mM IPTG for 18 hours at 18°C,which was 0.2 mM IPTG 6 hours at 30°C for ELK16 fusion hGH.The expression level of active aggregates was 44.9-158.3μg/mg cell wet weight,accounting for 29.5-86.7%of hGH fusion protein.2.8-21.4μg/mg cell wet weight of hGH were released into the supernatant by DTT induced self-cleavage in vitro,and total recovery of hGH ranging 9.1-30.2%.The highest yield of hGH was achieved by using L₆KD,which resulted in 21.4μg/mg wet cell weight,or 56.9 mg/L LB culture,and purity was estimated to be 88.2%.The mass spectrometric detection by UPLC-TOF/MS revealed that the purified hGH molecular mass is22678 Da,which is consistent with the theoretical value.The cell proliferation activity on Nb2-11of the purified hGH was 80%of that of commercial hGH.The ELISA assay showed that at the lowest concentration of 3.96 ng/mL,the purified hGH.And then,the SpyTag/SpyCatcher was introduced into cSAT scheme.However,the results shown that this modified scheme may not be applicable for hGH,IGF-1 and BNP.In this work,we approved a cSAT scheme for simple and effective production of active hGH in E.coli.