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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Bibliographic Details
Published in:Biology of reproduction 2000-03, Vol.62 (3), p.606-615
Main Authors: FURLONG, L. I, HELLMAN, U, KRIMER, A, TEZON, J. G, CHARREAU, E. H, VAZQUEZ-LEVIN, M. H
Format: Article
Language:English
Subjects:
Acrosin - genetics
Acrosin - immunology
Acrosin - metabolism
Biological and medical sciences
Enzyme Precursors - genetics
Enzyme Precursors - immunology
Enzyme Precursors - metabolism
Escherichia coli - genetics
Female
Fundamental and applied biological sciences. Psychology
Genetic Vectors
Humans
Immune Sera
Iodine Radioisotopes
Male
Mammalian male genital system
Morphology. Physiology
Protein Engineering - methods
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Analysis, Protein
Time Factors
Vertebrates: reproduction
Zona Pellucida - metabolism
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Summary: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 125 I-zona pellucida glycoproteins.
ISSN:0006-3363
1529-7268
DOI:10.1095/biolreprod62.3.606