Expression of mouse anticreatine kinase (MAK33) monoclonal antibody in the yeast Hansenula polymorpha.
The methylotrophic yeast Hansenula polymorpha HM1-39 (ura 3 and leu 2) was used as a host strain for the expression of the Fab fragment of the MAK33 monoclonal antibody. The MAK33 antibody reacts specifically with creatine kinase-M. The cDNA of kappa and gamma chains were inserted between the FMD or MOX promoter and the MOX terminator within the expression plasmids. In addition, the secretion signal sequence of the mating factor-alpha (prepro segment) and a fragment from glucoamylase with its secretion signal peptide, were also inserted in the expression plasmids for efficient secretion and production of the MAK33 monoclonal antibody. The co-expression of kappa and gamma chains was achieved by double transformation with kappa and then with gamma chain-expressing plasmids. The cells of H. polymorpha HM1-39 showed high mitotic stability and both uracil+ and leucine+ phenotypic stability after double transformation. Northern analysis showed a high rate of transcription of either kappa or gamma chain mRNA but not both, when the cells were grown in an induction medium. Protein analysis of double-transformed cells showed the monomers of the MAK33 antibody (kappa and gamma chains) were not assembled into a heterodimeric functional form. The expressed proteins of light and heavy chains represent about 11-12% of total cell protein and are found more inside than outside the cell. The expressed monomers show antigen-binding affinity in the Ouchterlony diffusion test; and the binding activity exhibited by cell-free extract was more than that of the cell culture supernatant.
Abdel-Salam
H A
HA
Department of Microbiology, Faculty of Pharmacy, Zagazig University, Egypt.
El-Khamissy
T
T
Enan
G A
GA
Hollenberg
C P
CP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015152:Blotting, Northern; D003402:Creatine Kinase / Q000276:immunology*; D007142:Immunoglobulin gamma-Chains / Q000096:biosynthesis; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis; D051379:Mice; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014170:Transformation, Genetic
D000911:Antibodies, Monoclonal; D007142:Immunoglobulin gamma-Chains; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; D003402:Creatine Kinase