A Novel Solid-Phase Site-Specific PEGylation Enhances the In Vitro and In Vivo Biostabilty of Recombinant Human Keratinocyte Growth Factor 1
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{"title"=>"A novel solid-phase site-specific pegylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1", "type"=>"journal", "authors"=>[{"first_name"=>"Zhifeng", "last_name"=>"Huang", "scopus_author_id"=>"55191568300"}, {"first_name"=>"Guanghui", "last_name"=>"Zhu", "scopus_author_id"=>"14122688100"}, {"first_name"=>"Chuanchuan", "last_name"=>"Sun", "scopus_author_id"=>"42361653000"}, {"first_name"=>"Jingui", "last_name"=>"Zhang", "scopus_author_id"=>"55205205100"}, {"first_name"=>"Yi", "last_name"=>"Zhang", "scopus_author_id"=>"55204934300"}, {"first_name"=>"Youting", "last_name"=>"Zhang", "scopus_author_id"=>"55204972300"}, {"first_name"=>"Chaohui", "last_name"=>"Ye", "scopus_author_id"=>"55702406700"}, {"first_name"=>"Xiaojie", "last_name"=>"Wang", "scopus_author_id"=>"55736998300"}, {"first_name"=>"Dariush", "last_name"=>"Ilghari", "scopus_author_id"=>"24474488400"}, {"first_name"=>"Xiaokun", "last_name"=>"Li", "scopus_author_id"=>"37761910800"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"364730689", "doi"=>"10.1371/journal.pone.0036423", "sgr"=>"84860501811", "scopus"=>"2-s2.0-84860501811", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"22574160"}, "id"=>"1a9f029f-95f1-385e-a41d-0da6eb57f18b", "abstract"=>"Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.", "link"=>"http://www.mendeley.com/research/novel-solidphase-sitespecific-pegylation-enhances-vitro-vivo-biostabilty-recombinant-human-keratinoc", "reader_count"=>17, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>1, "Researcher"=>5, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>2, "Student > Master"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>1, "Researcher"=>5, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>4, "Student > Postgraduate"=>2, "Student > Master"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>4, "Engineering"=>1, "Medicine and Dentistry"=>2, "Agricultural and Biological Sciences"=>8, "Sports and Recreations"=>1, "Chemical Engineering"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Sports and Recreations"=>{"Sports and Recreations"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>8}, "Unspecified"=>{"Unspecified"=>4}, "Chemical Engineering"=>{"Chemical Engineering"=>1}}, "reader_count_by_country"=>{"France"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/331411", "https://ndownloader.figshare.com/files/331470", "https://ndownloader.figshare.com/files/331523"], "description"=>"<div><p>Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the <em>in vitro</em> and <em>in vivo</em> biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl<sub>4</sub>-induced injury in rats compared to rhKGF-1.</p> </div>", "links"=>[], "tags"=>["solid-phase", "site-specific", "pegylation", "enhances", "biostabilty", "recombinant", "keratinocyte"], "article_id"=>125425, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.s001", "https://dx.doi.org/10.1371/journal.pone.0036423.s002", "https://dx.doi.org/10.1371/journal.pone.0036423.s003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Novel_Solid_Phase_Site_Specific_PEGylation_Enhances_the_In_Vitro_and_In_Vivo_Biostabilty_of_Recombinant_Human_Keratinocyte_Growth_Factor_1/125425", "title"=>"A Novel Solid-Phase Site-Specific PEGylation Enhances the <em>In Vitro</em> and <em>In Vivo</em> Biostabilty of Recombinant Human Keratinocyte Growth Factor 1", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-05-04 01:30:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/642129"], "description"=>"<p>Panel A. SDS-PAGE analysis of the fractions collected from Glutathione Sepharose chromatography. Lane M, molecular weight standards; lane 1, the cell lysate supernatant of BL21 (DE3) containing pET- GST-KGF-1 induced with 1.0 mM IPTG for 4 h; lane 2, the flow-through fraction; lane 3, the fraction eluted with 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0. Panel B. SDS-PAGE analysis of the fractions collected from CM Sepharose chromatography. Lane M, molecular weight standards; lane 4, the GST-rhKGF-1 fusion protein after treatment with thrombin; lane 5, the flow-through fraction; lane 6, NaCl-eluted fraction. Panel C. SDS-PAGE analysis of Heparin Sepharose chromatography. Lane M, molecular weight standards; lane 7, NaCl-eluted fraction from CM Sepharose Fast Flow column; lane 8, 0.45 M NaCl-eluted fraction; lane 8, 0.6 M NaCl-eluted fraction.</p>", "links"=>[], "tags"=>["purification"], "article_id"=>312618, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SDS_PAGE_analysis_of_expression_and_purification_of_rhKGF_1_/312618", "title"=>"SDS-PAGE analysis of expression and purification of rhKGF-1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:43:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/642199"], "description"=>"<p>Panel A. SDS-PAGE analysis of solid-phase PEGylated rhKGF-1. Lane M, molecular weight standards; lane a, solid-phase PEGylation products obtained at PEG-to-protein ratio of 10/1 and reaction time of 8 h; lane b, non-PEGylated rhKGF-1. Panel B. Elution profile of PEGylation reaction mixture on a SP Sepharose Fast Flow column. The Insert panel shows SDS-PAGE analysis of the fractions collected from SP Sepharose chromatography.</p>", "links"=>[], "tags"=>["elution", "solid-phase", "pegylation"], "article_id"=>312681, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SDS_PAGE_analysis_and_elution_profile_of_solid_phase_PEGylation_reaction_mixture_/312681", "title"=>"SDS-PAGE analysis and elution profile of solid-phase PEGylation reaction mixture.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:44:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/642281"], "description"=>"<p>Panel A, MALDI-TOF mass spectrometry of PEGylated rhKGF-1 showing the molecular mass of PEGylated rhKGF-1 (36997 Da). Panel B, MALDI-TOF mass spectrometry of non-PEGylated rhKGF-1 showing the molecular mass of non-PEGylated rhKGF-1 (16297 Da). Panel C, D. N-terminal sequencing of non-PEGylated and PEGylated rhKGF-1 by Edman degradation method.</p>", "links"=>[], "tags"=>["solid-phase", "pegylated"], "article_id"=>312763, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Validation_of_the_solid_phase_PEGylated_rhKGF_1_/312763", "title"=>"Validation of the solid-phase PEGylated rhKGF-1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:46:03"}
  • {"files"=>["https://ndownloader.figshare.com/files/642376"], "description"=>"<p>Panel A. NIH 3T3 fibroblasts were stimulated with rhKGF-1 or PEGylated rhKGF-1 at three does 1.5 µM, 3 µM, and 6 µM, and phosphorylation courses of ERK1/2 (p-ERK) were measured by immunoblotting alongside total ERK as loading controls. The blots shown are representative of three independent experiments; Panel B. Semi-quantitative analysis of the protein bands in Panel A. *, p<0.05 <i>vs</i> Normal control; <sup>#</sup>, p<0.05 <i>vs</i> the corresponding non-PEGylated rhKGF-1 group.</p>", "links"=>[], "tags"=>["rhkgf-1", "pegylation", "kinase"], "article_id"=>312853, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effect_of_rhKGF_1_PEGylation_on_MAP_kinase_activation_/312853", "title"=>"The effect of rhKGF-1 PEGylation on MAP kinase activation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:47:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/642457"], "description"=>"<p>Panel A. Far-UV CD spectra of non-PEGylated (black line) and solid-phase PEGylated rhKGF-1 (red line). The ellipticities are reported as mean residue ellipticity (MRE) (deg cm<sup>2</sup> dmol<sup>−1</sup>). Panel B. Effect of PEGyaltion on the proteolytic stability of rhKGF-1. PEGylated and intact rhKGF-1 were incubated with trypsin with a molar ratio of 10/1 (protein/trypsin) at 37°C for the indicated periods of times. Trypsin-treated rhKGF-1 was examined for the protein integrity by SDS-PAGE. The bands representing PEGylated and intact rhKGF-1 were quantified by densitometry scanning. The band densities of non-trypsin-treated intact and PEGylated rhKGF-1 are considered as 100% (indicated as control), while the band densities of trypsin-treated PEGylated or intact rhKGF-1 are presented as relative percentage to the control. *, p<0.05 <i>vs</i> the corresponding intact rhKGF-1 group. Panel C. Kinetic plots of remaining soluble non-PEGylated rhKGF-1 (black square) and PEGylated rhKGF-1 (red circle). The soluble protein concentration was determined by Bicinchoninic acid (BCA) kit. Samples were first incubated at 37°C for 3 days and then transferred to 45°C for the rest of the experiments. Pane D. Effect of pH on <i>in vitro</i> bioactivity of non-PEGylated rhKGF-1 (black square) and PEGylated rhKGF-1 (red circle).</p>", "links"=>[], "tags"=>["solid-phase", "pegylation"], "article_id"=>312936, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_effect_of_solid_phase_PEGylation_on_the_structural_integrity_and_stability_of_rhKGF_1_/312936", "title"=>"The effect of solid-phase PEGylation on the structural integrity and stability of rhKGF-1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:48:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/642544"], "description"=>"<p>Panel A. Normal male SD rats were injected intravenously with 100 µg/kg rhKGF-1 (open circle), and PEGylated rhKGF-1 (solid circle). Blood samples were collected at the indicated time points. The amount of rhKGF-1 was measured by Human KGF-basic Mini ELISA Development Kit. A standard curve was made for each rhKGF-1, n = 6. Values are the mean±SD. Panel B. Comparison of half-life of non-PEGylated rhKGF-1 and PEGylated rhKGF-1. *, p<0.01 <i>vs</i> the corresponding non-PEGylated rhKGF-1 group.</p>", "links"=>[], "tags"=>["solid-phase", "pegylated", "rhkgf-1"], "article_id"=>313027, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pharmacokinetics_study_of_solid_phase_PEGylated_rhKGF_1_in_rats_/313027", "title"=>"Pharmacokinetics study of solid-phase PEGylated rhKGF-1 in rats.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:50:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/642627"], "description"=>"<p>48 male SD rats were randomly divided into four groups (12 rats/group): enalpril interventional group A (rhKGF-1-pretreated groups), enalpril interventional group B (PEGylated rhKGF-1-pretreated groups), injury-model group and healthy control group. The test groups were pretreated with non-PEGylated and PEGylated rhKGF-1 24 hours prior to 2.5 mL/kg CCl<sub>4</sub> administration. Four hours after the intraperitoneal injection of CCl<sub>4</sub>, rats were killed and their serum AST/ALT levels were determined. *, p<0.05 <i>vs</i> control group; <sup>#</sup>, p<0.05 <i>vs</i> injury-model group; <sup>ss</sup>, p<0.05 between indicated groups.</p>", "links"=>[], "tags"=>["solid-phase", "pegylated", "rhkgf-1", "serum", "ast", "alt", "levels", "rats", "intoxicated"], "article_id"=>313099, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_solid_phase_PEGylated_rhKGF_1_on_serum_AST_and_ALT_levels_in_rats_intoxicated_with_CCl_4_/313099", "title"=>"Effect of solid-phase PEGylated rhKGF-1 on serum AST and ALT levels in rats intoxicated with CCl<sub>4</sub>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-05-04 00:51:39"}
  • {"files"=>["https://ndownloader.figshare.com/files/642714"], "description"=>"<p>Biological activity of native rhKGF-1 and PEGylated rhKGF-1.</p>", "links"=>[], "tags"=>["rhkgf-1", "pegylated"], "article_id"=>313197, "categories"=>["Chemistry", "Developmental Biology", "Biochemistry", "Pharmacology", "Physiology"], "users"=>["Zhifeng Huang", "Guanghui Zhu", "Chuanchuan Sun", "Jingui Zhang", "Yi Zhang", "Youting Zhang", "Chaohui Ye", "Xiaojie Wang", "Dariush Ilghari", "Xiaokun Li"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0036423.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Biological_activity_of_native_rhKGF_1_and_PEGylated_rhKGF_1_/313197", "title"=>"Biological activity of native rhKGF-1 and PEGylated rhKGF-1.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-05-04 00:53:17"}

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Relative Metric

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