Expression of human proacrosin in Escherichia coli and binding to zona pellucida
Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity. The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression...
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todo:paper_00063363_v62_n3_p606_Furlong2023-10-03T14:04:50Z Expression of human proacrosin in Escherichia coli and binding to zona pellucida Furlong, L.I. Hellman, U. Krimer, A. Tezón, J.G. Charreau, E.H. Vazquez-Levin, M.H. complementary DNA epitope glycoprotein iodine 125 polyclonal antibody proacrosin recombinant protein article controlled study DNA sequence Escherichia coli gene expression human human cell male nonhuman polyacrylamide gel electrophoresis polymerase chain reaction priority journal protein expression zona pellucida Escherichia coli Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity. The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression of the full-length fusion protein was not detected. In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42-44 kDa) was recognized by a monoclonal antibody to human acrosin, AcrC5F10. A 32-34-kDa protein (Rec-30), not recognized by AcrC5F10 on Western blots, was the major expression product. Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression products as were Rec-40 and Rec-30, and truncated products from the C terminus were detected in the soluble fraction. Rec-40 and Rec-30 coexisted at any culture time tested. Immune serum raised against Rec-30 (AntiRec-30) stained the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts. Amino acid sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-terminal fragments of proacrosin. The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous 125I-zona pellucida glycoproteins. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00063363_v62_n3_p606_Furlong |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
complementary DNA epitope glycoprotein iodine 125 polyclonal antibody proacrosin recombinant protein article controlled study DNA sequence Escherichia coli gene expression human human cell male nonhuman polyacrylamide gel electrophoresis polymerase chain reaction priority journal protein expression zona pellucida Escherichia coli |
| spellingShingle |
complementary DNA epitope glycoprotein iodine 125 polyclonal antibody proacrosin recombinant protein article controlled study DNA sequence Escherichia coli gene expression human human cell male nonhuman polyacrylamide gel electrophoresis polymerase chain reaction priority journal protein expression zona pellucida Escherichia coli Furlong, L.I. Hellman, U. Krimer, A. Tezón, J.G. Charreau, E.H. Vazquez-Levin, M.H. Expression of human proacrosin in Escherichia coli and binding to zona pellucida |
| topic_facet |
complementary DNA epitope glycoprotein iodine 125 polyclonal antibody proacrosin recombinant protein article controlled study DNA sequence Escherichia coli gene expression human human cell male nonhuman polyacrylamide gel electrophoresis polymerase chain reaction priority journal protein expression zona pellucida Escherichia coli |
| description |
Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and assesses zona pellucida binding activity. The cDNA encoding human proacrosin was subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression of the full-length fusion protein was not detected. In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42-44 kDa) was recognized by a monoclonal antibody to human acrosin, AcrC5F10. A 32-34-kDa protein (Rec-30), not recognized by AcrC5F10 on Western blots, was the major expression product. Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression products as were Rec-40 and Rec-30, and truncated products from the C terminus were detected in the soluble fraction. Rec-40 and Rec-30 coexisted at any culture time tested. Immune serum raised against Rec-30 (AntiRec-30) stained the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts. Amino acid sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-terminal fragments of proacrosin. The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous 125I-zona pellucida glycoproteins. |
| format |
JOUR |
| author |
Furlong, L.I. Hellman, U. Krimer, A. Tezón, J.G. Charreau, E.H. Vazquez-Levin, M.H. |
| author_facet |
Furlong, L.I. Hellman, U. Krimer, A. Tezón, J.G. Charreau, E.H. Vazquez-Levin, M.H. |
| author_sort |
Furlong, L.I. |
| title |
Expression of human proacrosin in Escherichia coli and binding to zona pellucida |
| title_short |
Expression of human proacrosin in Escherichia coli and binding to zona pellucida |
| title_full |
Expression of human proacrosin in Escherichia coli and binding to zona pellucida |
| title_fullStr |
Expression of human proacrosin in Escherichia coli and binding to zona pellucida |
| title_full_unstemmed |
Expression of human proacrosin in Escherichia coli and binding to zona pellucida |
| title_sort |
expression of human proacrosin in escherichia coli and binding to zona pellucida |
| url |
http://hdl.handle.net/20.500.12110/paper_00063363_v62_n3_p606_Furlong |
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| _version_ |
1807317115196145664 |