{"id":{"repo_id":"ohiolink","oai_identifier":"oai:etd.ohiolink.edu:toledo1365202687"},"canonical_url":"https://search.dev.ndltd.org/etd/ohiolink/oai:etd.ohiolink.edu:toledo1365202687","repository":{"repo_id":"ohiolink","name":"OhioLINK","base_url":"https://etd.ohiolink.edu/acprod/odb_etd/ws/oai/oai"},"display":{"title":"Cloning, Expression and Purification of the Different Human Haptoglobin Chains and Initial Characterization by Mass Spectrometry","abstract":"Haptoglobin (Hp) is an acute phase glycoprotein that exists in all vertebrates and can be found in the majority of body fluids. It is phenotypically variant in humans and presents in two allelic forms: Hp1 and Hp2. The two forms vary by the identity of their light chains: L1 or L2 for Hp1 and Hp2, respectively. Hp is most well known for its function in rapidly binding to free hemoglobin (Hb), which is subsequently removed from circulation, making this protein very important in the prevention of Hb-based oxidative damage and a natural bacteriostat. Despite much research efforts in the past few decades, there is little information available on the structure of human Hp and its complex with Hb. This is in large part attributed to its large size, high degree of glycosylation, structural heterogeneity, and difficulty to obtain (due to it being an acute phase protein). Recombinantly producing Hp and its individual chains in E. coli solves all of these problems. This thesis presents the cloning, expression, purification, and initial characterization of the individual chains of human Hp: L1, L2, and H. The cloning, expression and purification of L1 and L2 were successful. Correct protein sequences were confirmed using trypsin in-gel digestion, MALDI-TOF mass spectrometry, and tandem mass spectrometry. Cloning for the H-chain is ongoing as of the publication date of this thesis. Using chemical modification by iodoacetamide and charge state distributions in ESI mass spectra, results support L1 contained the correct disulfide pattern, while L2 did not. In order to obtain the proper folding and the correct disulfide pattern for L2, it will be necessary to introduce a renaturation step.In addition to the Hp project, this thesis included work done to elucidate the regions of disorder in the protein DnaT. DnaT is one of the primosomal proteins that is an essential component of the PriA replication restart pathway. Very little is known about this protein, its structure, and its actual mode of function. Several attempts to crystallize this protein have been met with inconclusive results thought to be due to regions of intrinsic disorder in the protein and degradation during the crystallization process. We were able to identify the cleavage sites in the degradation of the protein by sequence analysis and ESI MS of fresh and degraded protein samples. Using analysis of the charge state distributions in ESI MS, we provide definitive evidence of the intrinsic disorder of E. coli DnaT. Through collision-induced dissociations studies and the top-down approach, we were able to identify that the N-terminal part of the protein was the flexible regions. This allowed the proposal of a sequence construct lacking this flexible region for future crystallization studies.","abstract_html":"Haptoglobin (Hp) is an acute phase glycoprotein that exists in all vertebrates and can be found in the majority of body fluids. It is phenotypically variant in humans and presents in two allelic forms: Hp1 and Hp2. The two forms vary by the identity of their light chains: L1 or L2 for Hp1 and Hp2, respectively. Hp is most well known for its function in rapidly binding to free hemoglobin (Hb), which is subsequently removed from circulation, making this protein very important in the prevention of Hb-based oxidative damage and a natural bacteriostat. Despite much research efforts in the past few decades, there is little information available on the structure of human Hp and its complex with Hb. This is in large part attributed to its large size, high degree of glycosylation, structural heterogeneity, and difficulty to obtain (due to it being an acute phase protein). Recombinantly producing Hp and its individual chains in E. coli solves all of these problems. This thesis presents the cloning, expression, purification, and initial characterization of the individual chains of human Hp: L1, L2, and H. The cloning, expression and purification of L1 and L2 were successful. Correct protein sequences were confirmed using trypsin in-gel digestion, MALDI-TOF mass spectrometry, and tandem mass spectrometry. Cloning for the H-chain is ongoing as of the publication date of this thesis. Using chemical modification by iodoacetamide and charge state distributions in ESI mass spectra, results support L1 contained the correct disulfide pattern, while L2 did not. In order to obtain the proper folding and the correct disulfide pattern for L2, it will be necessary to introduce a renaturation step.In addition to the Hp project, this thesis included work done to elucidate the regions of disorder in the protein DnaT. DnaT is one of the primosomal proteins that is an essential component of the PriA replication restart pathway. Very little is known about this protein, its structure, and its actual mode of function. Several attempts to crystallize this protein have been met with inconclusive results thought to be due to regions of intrinsic disorder in the protein and degradation during the crystallization process. We were able to identify the cleavage sites in the degradation of the protein by sequence analysis and ESI MS of fresh and degraded protein samples. Using analysis of the charge state distributions in ESI MS, we provide definitive evidence of the intrinsic disorder of E. coli DnaT. Through collision-induced dissociations studies and the top-down approach, we were able to identify that the N-terminal part of the protein was the flexible regions. This allowed the proposal of a sequence construct lacking this flexible region for future crystallization studies.","abstract_has_math":false,"creators":["Lombard, Camille"],"institution":"University of Toledo","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Griffith, Wendell"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2013,"date_issued":"2013-06-14","date_published":"2013-06-14","updated_at":"2026-07-24T03:37:31Z","subjects":["Chemistry","Human haptoglobin","cloning","mass spectrometry","DnaT","IMMS"],"languages":["English"],"rights":["unrestricted","This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://rave.ohiolink.edu/etdc/view?acc_num=toledo1365202687","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Griffith, Wendell"]},{"key":"dc:creator","label":"Author","values":["Lombard, Camille"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2013-06-14"]},{"key":"dc:publisher","label":"Institution","values":["University of Toledo / OhioLINK"]},{"key":"dc:type","label":"Dc Type","values":["Electronic Thesis or Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Toledo"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Chemistry","Human haptoglobin","cloning","mass spectrometry","DnaT","IMMS"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English"]},{"key":"dc:rights","label":"Dc Rights","values":["unrestricted","This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://rave.ohiolink.edu/etdc/view?acc_num=toledo1365202687"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Haptoglobin (Hp) is an acute phase glycoprotein that exists in all vertebrates and can be found in the majority of body fluids. It is phenotypically variant in humans and presents in two allelic forms: Hp1 and Hp2. The two forms vary by the identity of their light chains: L1 or L2 for Hp1 and Hp2, respectively. Hp is most well known for its function in rapidly binding to free hemoglobin (Hb), which is subsequently removed from circulation, making this protein very important in the prevention of Hb-based oxidative damage and a natural bacteriostat. Despite much research efforts in the past few decades, there is little information available on the structure of human Hp and its complex with Hb. This is in large part attributed to its large size, high degree of glycosylation, structural heterogeneity, and difficulty to obtain (due to it being an acute phase protein). Recombinantly producing Hp and its individual chains in E. coli solves all of these problems. This thesis presents the cloning, expression, purification, and initial characterization of the individual chains of human Hp: L1, L2, and H. The cloning, expression and purification of L1 and L2 were successful. Correct protein sequences were confirmed using trypsin in-gel digestion, MALDI-TOF mass spectrometry, and tandem mass spectrometry. Cloning for the H-chain is ongoing as of the publication date of this thesis. Using chemical modification by iodoacetamide and charge state distributions in ESI mass spectra, results support L1 contained the correct disulfide pattern, while L2 did not. In order to obtain the proper folding and the correct disulfide pattern for L2, it will be necessary to introduce a renaturation step.In addition to the Hp project, this thesis included work done to elucidate the regions of disorder in the protein DnaT. DnaT is one of the primosomal proteins that is an essential component of the PriA replication restart pathway. Very little is known about this protein, its structure, and its actual mode of function. Several attempts to crystallize this protein have been met with inconclusive results thought to be due to regions of intrinsic disorder in the protein and degradation during the crystallization process. We were able to identify the cleavage sites in the degradation of the protein by sequence analysis and ESI MS of fresh and degraded protein samples. Using analysis of the charge state distributions in ESI MS, we provide definitive evidence of the intrinsic disorder of E. coli DnaT. Through collision-induced dissociations studies and the top-down approach, we were able to identify that the N-terminal part of the protein was the flexible regions. This allowed the proposal of a sequence construct lacking this flexible region for future crystallization studies."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf","p.184","7.29 MB"]},{"key":"dc:title","label":"Title","values":["Cloning, Expression and Purification of the Different Human Haptoglobin Chains and Initial Characterization by Mass Spectrometry"]}]}],"canonical_facts":{"dc:contributor":["Griffith, Wendell"],"dc:creator":["Lombard, Camille"],"dc:date":["2013-06-14"],"dc:description":["Haptoglobin (Hp) is an acute phase glycoprotein that exists in all vertebrates and can be found in the majority of body fluids. It is phenotypically variant in humans and presents in two allelic forms: Hp1 and Hp2. The two forms vary by the identity of their light chains: L1 or L2 for Hp1 and Hp2, respectively. Hp is most well known for its function in rapidly binding to free hemoglobin (Hb), which is subsequently removed from circulation, making this protein very important in the prevention of Hb-based oxidative damage and a natural bacteriostat. Despite much research efforts in the past few decades, there is little information available on the structure of human Hp and its complex with Hb. This is in large part attributed to its large size, high degree of glycosylation, structural heterogeneity, and difficulty to obtain (due to it being an acute phase protein). Recombinantly producing Hp and its individual chains in E. coli solves all of these problems. This thesis presents the cloning, expression, purification, and initial characterization of the individual chains of human Hp: L1, L2, and H. The cloning, expression and purification of L1 and L2 were successful. Correct protein sequences were confirmed using trypsin in-gel digestion, MALDI-TOF mass spectrometry, and tandem mass spectrometry. Cloning for the H-chain is ongoing as of the publication date of this thesis. Using chemical modification by iodoacetamide and charge state distributions in ESI mass spectra, results support L1 contained the correct disulfide pattern, while L2 did not. In order to obtain the proper folding and the correct disulfide pattern for L2, it will be necessary to introduce a renaturation step.In addition to the Hp project, this thesis included work done to elucidate the regions of disorder in the protein DnaT. DnaT is one of the primosomal proteins that is an essential component of the PriA replication restart pathway. Very little is known about this protein, its structure, and its actual mode of function. Several attempts to crystallize this protein have been met with inconclusive results thought to be due to regions of intrinsic disorder in the protein and degradation during the crystallization process. We were able to identify the cleavage sites in the degradation of the protein by sequence analysis and ESI MS of fresh and degraded protein samples. Using analysis of the charge state distributions in ESI MS, we provide definitive evidence of the intrinsic disorder of E. coli DnaT. Through collision-induced dissociations studies and the top-down approach, we were able to identify that the N-terminal part of the protein was the flexible regions. This allowed the proposal of a sequence construct lacking this flexible region for future crystallization studies."],"dc:format":["application/pdf","p.184","7.29 MB"],"dc:identifier":["http://rave.ohiolink.edu/etdc/view?acc_num=toledo1365202687"],"dc:language":["English"],"dc:publisher":["University of Toledo / OhioLINK"],"dc:rights":["unrestricted","This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws."],"dc:subject":["Chemistry","Human haptoglobin","cloning","mass spectrometry","DnaT","IMMS"],"dc:title":["Cloning, Expression and Purification of the Different Human Haptoglobin Chains and Initial Characterization by Mass Spectrometry"],"dc:type":["Electronic Thesis or Dissertation"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["University of Toledo"]},"updated_at":"2026-07-24T03:37:31Z"}