Production of Biologically Active Recombinant Human Lactoferrin in Aspergillus Oryzae

We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae α–amylase promoter and the 3′ flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron–binding capacity. The recombinant protein appears to be appropriately N–linked glycosylated and correctly processed at the N–terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large–scale production and secretion of biologically active mammalian glycoproteins.

[1]  J. Brunt.  Fungi: The Perfect Hosts? , 1986, Bio/Technology.

[2]  R. Berka,et al.  Improved Production of Chymosin in Aspergillus by Expression as a Glucoamylase-Chymosin Fusion , 1990, Bio/Technology.

[3]  Edward N. Baker,et al.  Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins , 1990, Nature.

[4]  M. Tuite,et al.  Heterologous gene expression in filamentous fungi , 1989 .

[5]  N. Pieniązek,et al.  4-nitroquinoline-1-oxide: A good mutagen for Aspergillus nidulans , 1977 .

[6]  L. Thim,et al.  High Level Expression of Recombinant Genes in Aspergillus Oryzae , 1988, Bio/Technology.

[7]  J. Heremans,et al.  An iron-binding protein common to many external secretions , 1966 .

[8]  P. Ralph,et al.  Specificity and modulation of the action of lactoferrin, a negative feedback regulator of myelopoiesis. , 1980, Blood.

[9]  H. Murakami,et al.  Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium. , 1983, Biochimica et biophysica acta.

[10]  K. Német,et al.  The biological role of lactoferrin. , 1985, Haematologia.

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

[12]  A. Means,et al.  Characterization and expression of the unique calmodulin gene of Aspergillus nidulans. , 1990, The Journal of biological chemistry.

[13]  R. Berka,et al.  Commercial Levels of Chymosin Production by Aspergillus , 1991, Bio/Technology.

[14]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[15]  E. Benz,et al.  Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis. , 1987, Blood.

[16]  M. Groves.  The isolation of a red protein from milk. , 1960 .

[17]  B. Coddeville,et al.  Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species. , 1988, Biochimie.

[18]  R. Waring,et al.  Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. , 1989, Gene.

[19]  L. Hnilica,et al.  Lactoferrin: nuclear localization in the human neutrophilic granulocyte? , 1981, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[20]  F. Schoentgen,et al.  Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins. , 1984, European journal of biochemistry.

[21]  E. Boel,et al.  Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger. , 1984, The EMBO journal.

[22]  J. Mcghee,et al.  A bactericidal effect for human lactoferrin. , 1977, Science.

[23]  J. Brunt,et al.  A Direct Route to Biotech Directories , 1986, Bio/Technology.

[24]  V. Seligy,et al.  Aspergillus oryzae has two nearly identical Taka-amylase genes, each containing eight introns. , 1989, Gene.

[25]  D. Rice,et al.  Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. , 1989, Journal of molecular biology.

[26]  L. Boxer,et al.  Lactoferrin: a promoter of polymorphonuclear leukocyte adhesiveness. , 1981, Blood.

[27]  S. Osmani,et al.  Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans , 1987, The Journal of cell biology.

[28]  W. Funk,et al.  Expression of cloned human lactoferrin in baby-hamster kidney cells. , 1991, The Biochemical journal.

[29]  M. Schlabach,et al.  The synergistic binding of anions and Fe3+ by transferrin. Implications for the interlocking sites hypothesis. , 1975, The Journal of biological chemistry.

[30]  F. Pieper,et al.  Generation Of Transgenic Dairy Cattle Using ‘in vitro’ Embryo Production , 1991, Bio/Technology.

[31]  V. Seligy,et al.  The induction of alpha-amylase by starch in Aspergillus oryzae: evidence for controlled mRNA expression. , 1984, Canadian journal of biochemistry and cell biology = Revue canadienne de biochimie et biologie cellulaire.

[32]  E. Schonne,et al.  LACTOFERRIN, AN IRON-BINBING PROTEIN NI NEUTROPHILIC LEUKOCYTES , 1969, The Journal of experimental medicine.

[33]  T. Yip,et al.  Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[34]  A. Tarentino,et al.  Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. , 1985, Biochemistry.

[35]  P. Vilja,et al.  A rapid and sensitive non-competitive avidin-biotin assay for lactoferrin. , 1985, Journal of immunological methods.

[36]  P. Thomas,et al.  Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. , 1980, Proceedings of the National Academy of Sciences of the United States of America.