Complementary deoxyribonucleic acid cloning and characterization of mSP-10: the mouse homologue of human acrosomal protein SP-10.
Complementary DNA encoding the putative mouse homologue for human acrosomal protein SP-10, a candidate contraceptive vaccinogen, was cloned and sequenced. The entire open reading frame (amino acids 18 to 261) of the mouse SP-10 (mSP-10), with the exception of the signal peptide (amino acids 1 to 17), was placed under the influence of inducible T7 RNA polymerase/promoter system to overproduce recombinant protein (re-mSP-10) in Escherichia coli. A six-histidine tag, which was coexpressed at the carboxyl terminus of re-mSP-10, provided the means for purification of re-mSP-10 by immobilized metal chelation affinity chromatography technique. The level of purity of re-mSP-10 thus obtained was determined by 2-dimensional gel electrophoresis to be 98%. Immunoblotting with monoclonal and polyclonal antibodies previously generated against human or baboon SP-10 showed that mSP-10 shared significant antigenic similarity with its primate counterparts. The position of mSP-10 in the mouse genome was next mapped through segregation analysis of an interspecific backcross panel of 96 animals. Acrv1 (assigned gene symbol for mSP-10) was localized in the proximal portion of mouse chromosome 9 in a region that exhibits synteny with human 11q23, the region to which ACRV1 (gene symbol for human SP-10) was previously mapped. These characterizations by combined immunological and gene mapping techniques established the cloned mSP-10 to be the mouse homologue of SP-10.
Reddi
P P
PP
Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Charlottesville, USA.
Naaby-Hansen
S
S
Aguolnik
I
I
Tsai
J Y
JY
Silver
L M
LM
Flickinger
C J
CJ
Herr
J C
JC
D000177:Acrosome; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics; D012739:Gonadal Steroid Hormones / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D017421:Sequence Analysis; D017385:Sequence Homology
C512729:ACRV1 protein, human; C080034:Acrv1 protein, mouse; D000941:Antigens; D018076:DNA, Complementary; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins; D011994:Recombinant Proteins