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Updated: Oct 5, 2026
Protein Engineering by Yeast Surface Display
Published on: November 29, 2024
Expression of mouse anticreatine kinase (MAK33) monoclonal antibody in the yeast Hansenula polymorpha
H A Abdel-Salam1, T El-Khamissy, G A Enan
1Department of Microbiology, Faculty of Pharmacy, Zagazig University, Egypt.
Abstract:
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.
Insights
This study expressed the MAK33 antibody in Hansenula polymorpha yeast, achieving high protein levels. However, the antibody
Area of Science:
- Biotechnology
- Molecular Biology
- Microbial Expression Systems
Background:
- The MAK33 monoclonal antibody targets creatine kinase-M.
- Hansenula polymorpha is a methylotrophic yeast utilized for recombinant protein production.
Purpose of the Study:
- To express the Fab fragment of the MAK33 monoclonal antibody in Hansenula polymorpha.
- To achieve efficient secretion and production of the antibody.
Main Methods:
- Utilized H. polymorpha HM1-39 as a host strain.
- Inserted cDNA for kappa and gamma chains into expression plasmids with FMD or MOX promoters.
- Employed secretion signal sequences from mating factor-alpha and glucoamylase.
- Co-expressed chains via double transformation.
- Analyzed transcription via Northern analysis and protein expression via protein analysis and Ouchterlony diffusion test.
Main Results:
- Achieved high mitotic and phenotypic stability in transformed H. polymorpha cells.
- High transcription rates for individual kappa or gamma chain mRNA were observed, but not both.
- Expressed monomers of MAK33 antibody (kappa and gamma chains) did not assemble into a functional heterodimeric form.
- Expressed proteins constituted 11-12% of total cell protein, primarily intracellular.
- Expressed monomers demonstrated antigen-binding affinity.
Conclusions:
- Hansenula polymorpha can efficiently express individual MAK33 antibody chains at high levels.
- The co-expression system did not result in the assembly of functional heterodimeric MAK33 antibody.
- Further optimization is needed to achieve functional antibody assembly in this yeast expression system.
