Preparation And Characterization Of RNase-BSA Conjugates Mediated By PEG

Bioconjugation technology is the process of conjugate proteins with proteins or otherbiomolecules by chemical crosslinking and modification methods, to alter the solubility or otherproperties of the original protein. Conjugation of albumin to the protein drug is achieved bycovalent bond that can be broken by the enzyme in vivo, which imparts resistance to proteolysis,improves solubility and achieves targeting. Poly-ethylene glycol (PEG) has been widely used infood, medicine, cosmetics, chemical fiber and other fields, due to its good water-solubility andfavorable compatibility with many organic components. PEGylation can improve the therapeuticefficacy of proteins by enhancing persistence in circulation and reducing immunogenicity andantigenicity. The structure and function of bovine pancreatic ribonuclease A (RNase A) has beenclearly known. Recent studies have found that the super-family and structural analogues haveanti-tumor activity. However, its own performance is not obvious. To strengthen and stabilize thebiological activity, RNase A was chosen as the model protein in the present study. Theconjugation of RNase A with albumin (BSA) by crosslinkers like PEG was conducted.First, the RNase-BSA conjugates were prepared. Heterobifunctional crosslinkersSulfo-SMCC, PEG3500and assistant crosslinker Hexadecylamine (HDA) were chosen tofacilitate conjugation of RNase to albumin. The optimal conjugation conditions were analyzedby size exclusion chromatography using analytical grade Superdex200column (1cm×30cm)and SDS-PAGE. The result showed that the optimal reactant ratios were:BSA:NEM:SMCC:RNase:IT=1:5:20:8:80; BSA:IT:RNase:PEG3500=1:10:0.8:4;RNase:IT:PEG3500:HDA=1:3:2:2,20μL of0.2mmol/L BSA was added to200μL reactionsystem, and all reactions were taken at4℃. AKTA Purification System and preparative gradeSuperdex200(2.6cm×70cm) were used for purification of the RNase-BSA conjugates.Secondly, the structural and functional characterization of the RNase-BSA conjugates wascarried out. Structural characterization of the conjugates was carried out using intrinsicfluorescence spectra, dynamic light scattering (DLS) and sedimentation velocity analyticalultracentrifugation (AUC). The present study suggested that compared with the weak emissionfluorescence intensity of RNase A which was equivalent to a quencher, BSA was predominant. The hydrodynamic radius of RNase A was increased greatly after conjugation of albumin. Whilethe RNase-SMCC-BSA conjugate showed the largest sedimentation coefficients (S20, w) andlowest frictional ratios (f/f0). It was presumably related to the molecular weight, the properties ofcrosslinkers and the structural characteristic of the conjugates. Then, the result of vitro activityshowed that the bioactivity of RNase-SMCC-BSA conjugate was the highest, and the bioactivityof75%was remained overnight (above19h), delay releasing was realized. This indicated that thealbumin binding and PEGylation would be a better strategy to improve the therapeutic efficacyof RNase A.The present strategy may rationally optimize and tailor the pharmacological properties ofpeptides and proteins. Moreover, it can also provide a reference for application in many fieldslike food, feed, cosmetics, medicine and so on.