Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris.
Recombinant porcine lactoferrin (rPLF) was synthesized in Pichia pastoris using a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene. Strains expressing rPLF with its own signal sequence or with that from the yeast alpha-mating factor (alpha-MF) were able to produce and secrete rPLF, but levels were consistently higher using alpha-MF constructs. In contrast, P. pastoris strains that expressed rPLF without a signal sequence produced the protein in an insoluble intracellular form. Increasing the initial pH of shake-flask culture medium from 6.0 to 7.0 or adding ferric ions to the medium (to 100 microM) resulted in significant improvements in expression of rPLF from P. pastoris. Expression levels (approximately 12 mg/L) were much higher than those observed from Saccharomyces cerevisiae strains (1-2 mg/L). P. pastoris-secreted rPLF was isolated and purified via a one-step simple procedure using a heparin column. The molecular size (78 kDa), isoelectric point (8.8-9.0), N-terminal amino acid sequence, and iron-binding capability of rPLF were each similar to that of native milk PLF.
Wang
Sue-Hong
SH
Jen-Te Junior College of Medicine Nursing and Management, Miaoli, Taiwan.
Yang
Tien-Shuh
TS
Lin
Shiang-Ming
SM
Tsai
Ming-Shiun
MS
Wu
Shinn-Chih
SC
Mao
Simon J T
SJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005800:Genes, Fungal; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007781:Lactoferrin / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013997:Time Factors
D011994:Recombinant Proteins; D007501:Iron; D007781:Lactoferrin