title: Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization.
abstract: Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.
authors.affiliation: Institute of Immunological Engineering, Lyubuchany, Moscow Region, Chekhov District 142380, Russia. elena_dudich@mail.ru
authors.forename: Elena
authors.initials: E
authors.lastname: Dudich
authors.forename: Igor
authors.initials: I
authors.lastname: Dudich
authors.forename: Lidia
authors.initials: L
authors.lastname: Semenkova
authors.forename: Sergey
authors.initials: S
authors.lastname: Benevolensky
authors.forename: Elena
authors.initials: E
authors.lastname: Morozkina
authors.forename: Aleksey
authors.initials: A
authors.lastname: Marchenko
authors.forename: Sergey
authors.initials: S
authors.lastname: Zatcepin
authors.forename: Dmitry
authors.initials: D
authors.lastname: Dudich
authors.forename: Galina
authors.initials: G
authors.lastname: Soboleva
authors.forename: Luidmila
authors.initials: L
authors.lastname: Khromikh
authors.forename: Olga
authors.initials: O
authors.lastname: Roslovtceva
authors.forename: Eduard
authors.initials: E
authors.lastname: Tatulov
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002942:Circular Dichroism; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013696:Temperature; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: C097353:AFP protein, human; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins