Molecular cloning of cDNA for human prostatic acid phosphatase

Lee Chuan C. Yeh, Andrew J. Lee, Nathan E. Lee, Kwok Wai Lam, John C. Lee

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8-2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.

Original languageEnglish (US)
Pages (from-to)191-196
Number of pages6
JournalGene
Volume60
Issue number2-3
DOIs
StatePublished - 1987

Keywords

  • E. coli lysogen
  • Recombinant DNA
  • fusion protein
  • human gene library
  • phage λ gtll

ASJC Scopus subject areas

  • Genetics

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