Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.

[1]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[2]  B. Vogelstein,et al.  Preparative and analytical purification of DNA from agarose. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[3]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[4]  T. Saito,et al.  An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. , 1982, European journal of biochemistry.

[5]  H. Matsubara,et al.  High recovery of tryptophan from acid hydrolysates of proteins. , 1969, Biochemical and biophysical research communications.

[6]  J. Messing,et al.  Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites , 1978, Nature.

[7]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[8]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[9]  T. Narikawa,et al.  Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. , 1988, Biochimica et biophysica acta.

[10]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[11]  D. Helfman,et al.  Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[12]  H. Neu,et al.  The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. , 1965, The Journal of biological chemistry.

[13]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. Chambon,et al.  A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. , 1981, Analytical biochemistry.

[15]  K. Miura,et al.  PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. , 1963, Biochimica et biophysica acta.

[16]  M Mandel,et al.  Calcium-dependent bacteriophage DNA infection. , 1970, Journal of molecular biology.

[17]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[18]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[19]  S. Henikoff Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. , 1984, Gene.

[20]  T. Narikawa,et al.  Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1. , 1986, Biochimica et biophysica acta.

[21]  D. W. Ribbons,et al.  SOME ASPECTS OF THE ENDOGENOUS METABOLISM OF BACTERIA. , 1964, Bacteriological reviews.

[22]  S. Horinouchi,et al.  A New Isolation Method of Plasmid Deoxyribonucleic Acid from Staphylococcus aureus Using a Lytic Enzyme of Achromobacter lyticus , 1977 .

[23]  L. Heppel,et al.  A study of the substrate specificity and other properties of the alkaline phosphatase of Escherichia coli. , 1962, The Journal of biological chemistry.