Advances in peptidyl Asx-specific ligases for the application of cyclic peptides / 药学学报

Asparaginyl endopeptidases (AEPs) in plants belong to the family of cysteine protease that undergo self-activation in the form of zymogen in acidic vacuole and play important physiological roles in maturation of seed storage proteins, protein degradation, programmed cell death and host defense. Bioprocessing enzymes (peptidyl Asx-specific ligases, PALs) that promote the maturation of cyclotides have recently been isolated and identified from several cyclotide-rich plants. PALs derived from AEPs can site-specifically catalyze the formation of asparagine or aspartate peptide bonds. Due to the advantages of relatively traceless peptide bonds and broad substrate spectrum and high catalytic efficiency, they have been playing important roles in the cyclization and modification of peptides and proteins, and are powerful tools for improving the stability of peptide drugs. This review describes the physiological functions of AEPs in plants and summarizes the discoveries, structural characteristics, catalytic mechanism and protein engineering of PALs, as well as the limitation of their applications and future trends. In addition, the applications of PALs in cyclotides biosynthesis and the development of macrocyclic peptides are highlighted, with the aim of providing a new idea for the biocatalytic synthesis of cyclic peptides.

Development of a radiolabeled site-specific single-domain antibody positron emission tomography probe for monitoring PD-L1 expression in cancer / 药物分析学报

Despite advances in immunotherapy for the treatment of cancers,not all patients can benefit from programmed cell death ligand 1(PD-L1)immune checkpoint blockade therapy.Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level;hence,accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects.Therefore,based on the high affinity antibody Nb109,a new site-specifically radiolabeled tracer,68Ga-NODA-cysteine,aspartic acid,and valine(CDV)-Nb109,was designed and synthesized to accurately monitor PD-L1 expression.The tracer 68Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95％and radiochemical purity of 97％.It showed high affinity for PD-L1 with a dissociation constant of 12.34±1.65 nM.Both the cell uptake assay and positron emission tomography(PET)imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells.Meanwhile,dynamic PET imaging of a NC1-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression,allowing timely interventional immunotherapy.In conclusion,this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.

One-step synthesis of site-specific antibody-drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates

Glycosite-specific antibody‒drug conjugatess (gsADCs), harnessing Asn297 N-glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation process. Herein, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an endo-N-acetylglucosaminidase (ENGase) of Endo-S2. IgG glycoengineering via ENGases usually has two steps: deglycosylation by wild-type (WT) ENGases and transglycosylation by mutated ENGases. But in the current method, we have found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the one-step glycoengineering directly from native antibodies. Further studies on substrate specificity revealed that this approach has excellent tolerance on various modification of 6-Gal motif of LacNAc. Within 1 h, one-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups. These gsADCs demonstrated good homogeneity, buffer stability, in vitro and in vivo anti-tumor activity. This work presents a novel strategy using LacNAc-based substrates to reprogram the multi-step IgG glycoengineering to a one-step manner for highly efficient synthesis of gsADCs.

Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis / 生物工程学报

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome / 中国药科大学学报

@#Finding stable expression sites on the chromosomes of Chinese hamster ovary (CHO) cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture. Our group used lentiviral transfection to integrate the tracer gene (Zsgreen1) into the chromosome of CHO cells and found multiple potential stable expression sites. This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed, and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein (EGFP) gene into this site. Three strains of EGFP gene integrated cells were obtained. After 60 generations of suspension culture, the fluorescence intensity of the cells had no significant changes, which proved that this site can stably express the EGFP gene. The same method was used to construct recombinant CHO cell lines expressing the human serum albumin (HSA) gene, and was verified by Western blot that this site could express and secrete HSA. It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase / 生物工程学报

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

Site-specific Study on Warming Using Disposable Warmer “Kairo” in Healthy Subjects / 日本温泉気候物理医学会雑誌

  [Purpose] Acral coldness is a condition in which the fingers and/or toes are cold despite normal central body temperature. Regardless of the presence or absence of disease, many people suffer from acral coldness, especially elderly people. One method of relieving acral coldness is to heat the hands with a disposable warmer called “Kairo,” but this method interferes with daily work. We investigated whether or not heating other upper-limb parts with a pair of warmers can relieve acral coldness.   [Methods] After obtaining informed consent, 30 people who usually had acral coldness without other diseases received the Kairo holders for their necks, elbows, and wrists. Eighteen participants used a pair of Kairo warmers for each area for one week, respectively. The remaining participants wore only the holder to know the heat retention effect of the holders. All participants separately evaluated their acral coldness of the upper limbs and the cumbersomeness of holders and/or Kairo by using a 10cm visual analog scale (VAS). To measure the therapeutic effect of the Kairo, the temperature and blood flow of the surface of both hands were examined by thermography and laser speckle contrast analysis (LASCA) at the day after finishing each warming period. These evaluations were conducted after a 20-minute adaptation at 24°C.  [Result] The VAS of acral coldness of upper limbs decreased during the periods of warming on the neck and elbows with Kairo compared to the period without Kairo. There was no difference in the VAS value of the cumbersomeness among the neck, elbows, and wrists areas. The hand temperature on the day after the end of warming decreased slightly after elbow warming.  [Conclusion] We found that the coldness of the hand was improved by warming other parts, even if the hand was not warmed, but the effect varied depending on the warming area. No therapeutic effect was detected, but this was considered to be a problem in setting the measurement conditions. We believe these results will lead to new ways to improve acral coldness.

Site-specific Study on Warming Using Disposable Warmer “Kairo” in Healthy Subjects / 日本温泉気候物理医学会雑誌

  [Purpose] Acral coldness is a condition in which the fingers and/or toes are cold despite normal central body temperature. Regardless of the presence or absence of disease, many people suffer from acral coldness, especially elderly people. One method of relieving acral coldness is to heat the hands with a disposable warmer called “Kairo,” but this method interferes with daily work. We investigated whether or not heating other upper-limb parts with a pair of warmers can relieve acral coldness.   [Methods] After obtaining informed consent, 30 people who usually had acral coldness without other diseases received the Kairo holders for their necks, elbows, and wrists. Eighteen participants used a pair of Kairo warmers for each area for one week, respectively. The remaining participants wore only the holder to know the heat retention effect of the holders. All participants separately evaluated their acral coldness of the upper limbs and the cumbersomeness of holders and/or Kairo by using a 10cm visual analog scale (VAS). To measure the therapeutic effect of the Kairo, the temperature and blood flow of the surface of both hands were examined by thermography and laser speckle contrast analysis (LASCA) at the day after finishing each warming period. These evaluations were conducted after a 20-minute adaptation at 24°C.  [Result] The VAS of acral coldness of upper limbs decreased during the periods of warming on the neck and elbows with Kairo compared to the period without Kairo. There was no difference in the VAS value of the cumbersomeness among the neck, elbows, and wrists areas. The hand temperature on the day after the end of warming decreased slightly after elbow warming.  [Conclusion] We found that the coldness of the hand was improved by warming other parts, even if the hand was not warmed, but the effect varied depending on the warming area. No therapeutic effect was detected, but this was considered to be a problem in setting the measurement conditions. We believe these results will lead to new ways to improve acral coldness.

Molecular engineering of antibodies for site-specific conjugation to lipid polydopamine hybrid nanoparticles

Conjugation of antibodies to nanoparticles allows specific cancer targeting, but conventional conjugation methods generate heterogeneous conjugations that cannot guarantee the optimal orientation and functionality of the conjugated antibody. Here, a molecular engineering technique was used for site-specific conjugation of antibodies to nanoparticles. We designed an anti-claudin 3 (CLDN3) antibody containing a single cysteine residue, h4G3cys, then linked it to the maleimide group of lipid polydopamine hybrid nanoparticles (LPNs). Because of their negatively charged lipid coating, LPNs showed high colloidal stability and provided a functional surface for site-specific conjugation of h4G3cys. The activity of h4G3cys was tested by measuring the binding of h4G3cys-conjugated LPNs (C-LPNs) to CLDN3-positive tumor cells and assessing its subsequent photothermal effects. C-LPNsspecifically recognized CLDN3-overexpressing T47D breast cancer cells but not CLDN3-negative Hs578T breast cancer cells. High binding of C-LPNs to CLDN3-overexpressing T47D cells resulted in significantly higher temperature generation upon NIR irradiation and potent anticancer photothermal efficacy. Consistent with this, intravenous injection of C-LPNsin a T47D xenograft mouse model followed by NIR irradiation caused remarkable tumor ablation compared with other treatments through high temperature increases. Our results establish an accurate antibody-linking method and demonstrate the possibility of developing therapeutics using antibody-guided nanoparticles.

Updates on Approaches to Increase the Residence Time of Drug in the Stomach for Site Specific Delivery: Brief Review

In the field of oral drug delivery system, a gastroretentive system is gaining popularity day by day. Numerous of research work and extensive literature are published in past few years on gastroretentive drug delivery system. It is the one of the best and appropriate approaches for increasing the residence time of drug in the stomach and diffuses drug slowly in the sustained manner which helps in the site-specific delivery of the drug as well also increases the bioavailability at site-specific of delivery. This helps in many challenges associated with conventional oral drug delivery system. Different ways are used for approaching gastroretention viz. swelling and expandable system, high-density system, magnetic system, bioadhesive system and buoyant system with or without gas generating agents. During data mining well in vitro characterization and in vivo characterization including gamma scintigraphic and MRI techniques are well established and reported. But, still, today in vivo characterization technique is major challenging for the researcher due to its limitation. The documented literature explains the use of animal models like beagle dogs, rabbits and human subjects for in vivo evaluation parameter but it leads to increase in variation that抯 why this delivery system is limited in the market. This paper contains the latest literature compilation and various techniques used for gastroretention with its pros and cons. This review paper helps the researcher to take an overview of basics of gastroretentive drug delivery system and helps in understanding the basics of the system.

Protein PEGylation for the design of biobetters: from reaction to purification processes

The covalent attachment of polyethylene glycol (PEG) to therapeutical proteins is an important route to develop biobetters for biomedical, biotech and pharmaceutical industries. PEG conjugation can shield antigenic epitopes of the protein, reduce degradation by proteolytic enzymes, enhance long-term stability and maintain or even improve pharmacokinetic and pharmacodynamics characteristics of the protein drug. Nonetheless, correct information in terms of the PEGylation process from reaction to downstream processing is of paramount importance for the industrial application and processing scale-up. In this review we present and discuss the main steps in protein PEGylation, namely: PEGylation reaction, separation of the products and final characterization of structure and activity of the resulting species. These steps are not trivial tasks, reason why bioprocessing operations based on PEGylated proteins relies on the use of analytical tools according to the specific pharmaceutical conjugate that is being developed. Therefore, the appropriate selection of the technical and analytical methods may ensure success in implementing a feasible industrial process

MRI Features and Site-specific Factors of Ischemic Changes in White Matter: A Retrospective Study / 华中科技大学学报（医学）（英德文版）

Brain magnetic resonance imaging (MRI) of the elderly often reveals white matter changes (WMCs) with substantial variability across individuals.Our study was designed to explore MRI features and site-specific factors of ischemic WMCs.Clinical data of consecutive patients diagnosed with ischemic cerebral vascular disease who had undergone brain MRI were collected and analyzed.Multi-logistic regression analysis comparing patients with mild versus severe WMCs was performed to detect independent associations.Analyses of variance (ANOVAs) were used to detect regionally specific differences in lesions.We found that lesion distribution differed significantly across five cerebral areas,with lesions being predominant in the frontal lobe and parieto-occipital area.To explore WMCs risk factors,after adjusting for gender,diabetes mellitus,and hypertension,only age (P＜0.01),creatinine (P=0.01),alkaline phosphatase (ALP) (P=0.01) and low-density lipoprotein cholesterol (LDL-C) (P=0.03) were found to be independently associated with severe WMCs.Age (P＜0.001) was strongly associated with WMCs in the frontal lobe while hypertension was independently related to lesions in the basal ganglia (P=0.048) or infratentorial area (P=0.016).In conclusion,MRI of WMCs showed that ischemic WMCs occurred mostly in the frontal lobe and parieto-occipital area.The infratentorial area was least affected by WMCs.Typically,age-related WMCs were observed in the frontal lobes,while hypertension-related WMCs tended to occur in the basal ganglia and infratentorial area.

MRI Features and Site-specific Factors of Ischemic Changes in White Matter: A Retrospective Study / 华中科技大学学报（医学）（英德文版）

Brain magnetic resonance imaging (MRI) of the elderly often reveals white matter changes (WMCs) with substantial variability across individuals.Our study was designed to explore MRI features and site-specific factors of ischemic WMCs.Clinical data of consecutive patients diagnosed with ischemic cerebral vascular disease who had undergone brain MRI were collected and analyzed.Multi-logistic regression analysis comparing patients with mild versus severe WMCs was performed to detect independent associations.Analyses of variance (ANOVAs) were used to detect regionally specific differences in lesions.We found that lesion distribution differed significantly across five cerebral areas,with lesions being predominant in the frontal lobe and parieto-occipital area.To explore WMCs risk factors,after adjusting for gender,diabetes mellitus,and hypertension,only age (P＜0.01),creatinine (P=0.01),alkaline phosphatase (ALP) (P=0.01) and low-density lipoprotein cholesterol (LDL-C) (P=0.03) were found to be independently associated with severe WMCs.Age (P＜0.001) was strongly associated with WMCs in the frontal lobe while hypertension was independently related to lesions in the basal ganglia (P=0.048) or infratentorial area (P=0.016).In conclusion,MRI of WMCs showed that ischemic WMCs occurred mostly in the frontal lobe and parieto-occipital area.The infratentorial area was least affected by WMCs.Typically,age-related WMCs were observed in the frontal lobes,while hypertension-related WMCs tended to occur in the basal ganglia and infratentorial area.

Antibody-drug conjugates: recent advances in conjugation and linker chemistries

The antibody-drug conjugate (ADC), a humanized or human monoclonal antibody conjugated with highly cytotoxic small molecules (payloads) through chemical linkers, is a novel therapeutic format and has great potential to make a paradigm shift in cancer chemotherapy. This new antibody-based molecular platform enables selective delivery of a potent cytotoxic payload to target cancer cells, resulting in improved efficacy, reduced systemic toxicity, and preferable pharmacokinetics (PK)/pharmacodynamics (PD) and biodistribution compared to traditional chemotherapy. Boosted by the successes of FDA-approved Adcetris and Kadcyla, this drug class has been rapidly growing along with about 60 ADCs currently in clinical trials. In this article, we briefly review molecular aspects of each component (the antibody, payload, and linker) of ADCs, and then mainly discuss traditional and new technologies of the conjugation and linker chemistries for successful construction of clinically effective ADCs. Current efforts in the conjugation and linker chemistries will provide greater insights into molecular design and strategies for clinically effective ADCs from medicinal chemistry and pharmacology standpoints. The development of site-specific conjugation methodologies for constructing homogeneous ADCs is an especially promising path to improving ADC design, which will open the way for novel cancer therapeutics.

Design of recombinase and terminator-based genetic switches for cell state control / 生物工程学报

Various genetic switches have been developed to let engineered cells perform designed functions. However, a sustained input is often needed to maintain the on/off state, which is energy-consuming and sensitive to perturbation. Therefore, we developed a set of transcriptional switches for cell states control that were constructed by the inversion effect of site-specific recombinases on terminators. Such a switch could respond to a pulse signal and maintain the new state by itself until the next input. With a bottom-up design principle, we first characterized the terminators and recombinases. Then the mutual interference was studied to select compatible pairs, which were used to achieve one-time and two-time state transitions. Finally, we constructed a biological seven-segment display as a demonstration to prove such switch's immense potential for application.

Preparation of Site-specific Smart Nanocomposites for Drug Delivery and Study on Light-Controlled Cytarabine Release Performance / 中国药学杂志

OBJECTIVE: To prepare smart & site-specific drug carrier for controlled release purpose and study the bio-compatibilities and release performance.METHODS: By using high pressure thermo-heat method in autoclave, superparamagnetic core was obtained and further coated by SiO2 and MCM-41, therefore the “core-shell” structure was formed. To make the carrier “smart” and thus responsive to stimuli which was light in this research, the tunnels of the molecular sieve were grafted with gating molecules, 4,5-diazafluoren-9-one (indicated in the paper as DAFO). For bio-compatibilities testing, MTT in-vitro experiment was conducted. Cytarabine was used as test drug to preliminarily evaluate the controlled release performance of the drug carrier in vitro.RESULTS: The Fe3O4 nano-particles synthesized via high-pressure hydro-thermo procedure exhibited superparamagnetic with mean diameter of 280 nm. After SiO2 & molecular sieve coating steps and ligand grafting steps, the particles grew to 540 nm. The sub-structure of the carrier was confirmed by scanning/transmission electron microscope(SEM & TEM) and nitrogen adsorption/desorption. Our “smart” carrier was able to be guided to the sites or organs with magnetic field and more importantly it was able to unload drug molecules under 510 nm light irritation that could flip the gating-molecule. Furthermore, the drug carrier illustrated bio-compatibility and showed obvious cytotoxicity.CONCLUSION: The novel nanocomposites developed in this study can be used as targeted drug carrier.

Transglutaminase-based antibody-drug conjugation: antibody site-specific mutation and identification / 药学学报

Transglutaminase (TG) posttranslational modification of antibody permits more precisely conjugating. Based on the amino acid sequence of an anti-CD24 antibody (cG7), this article is aimed to generate a deglycosylated cG7 mutant (cG7Q). Firstly, we introduced additional glutamines at position 297 (N297Q) by site-directed mutagenesis, and then transfected the recombinant plasmids into CHO-s cells via electroporation method and screened by Dot blot assay. Subsequently, cG7Q was expressed and purified through Protein A affinity chromatography, further identified by SDS-PAGE electrophoresis and Western blot. Its affinity was detected with surface plasmon resonance and flow cytometry assay, and ADCC effect was determined by lactate dehydrogenase (LDH) release. Eventually, a cG7 mutant, cG7Q was successfully expressed with sequence-specific conjugation sites for further study.

Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector

Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.

Antibody-drug conjugates:design and clinical progress / 药学学报

Antibody-drug conjugates, constructed with monoclonal antibodies, linker and cytotoxins, have distinctive advantages over chemotherapy drugs and antibody drugs in cancer therapy. In this review, the strategy of developing ADCs, and the important progress in past decade are well summarized. The representative ADCs in the pipeline are introduced and characterized with their new features. While, perspective for future directions of ADCs is proposed.

Research on PEGylated uricase by periodate oxidation and reductive- amination / 中国药科大学学报

@#PEGylated uricase was prepared with the N-terminal amino site-specific modification by periodate oxidation followed by reductive-amination. A monomethoxy poly(ethylene glycol)intermediate was synthesized by amidation from monomethoxy poly(ethylene glycol)amine hydrochloride 20000(mPEG20000-NH2 ·HCl)with the relative molecular mass of 20 kD and N-(tert-butoxycarbonyl)-L-serine(Boc-Ser-OH), and then the Boc group of the intermediate was removed by trifluoroacetic acid(TFA)to produce the desired product Ser-mPEG20000. This compound could be oxidated by periodate to obtain a new poly(ethylene glycol)aldehyde derivative with high activity, which could be used to modify proteins with the N-terminal amino site-specific PEGylation after ultrafiltration, and the modification conditions to uricase by Ser-mPEG20000 were optimized. The structures of poly(ethylene glycol)intermediate and the target product were characterized by IR and 1H NMR, and the overall yield of the target product was 72. 8%. The preliminary modification to uricase indicated that the desired product Ser-mPEG20000 could modify proteins easily and efficiently. The optimal modification conditions of uricase PEGylated by Ser-mPEG20000 were obtained as follows: the molar ratio of Ser-mPEG20000 to uricase was 2 ∶1; the pH value of solution was 5. 0; the reaction temperature was 25 °C and the reaction time was 6 h.