Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Hansenula polymorpha is a yeast with a high growth rate and low nutrient requirements.
• It is widely recognized for its efficiency in expressing recombinant proteins.
• Methanol serves as an economical inducer for protein expression in this yeast.

Purpose of the Study:

• To construct and validate an expression vector for heterologous gene expression in Hansenula polymorpha.
• To achieve the expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha.
• To confirm the successful production and identity of recombinant GCSF.

Main Methods:

• A custom expression cassette was designed, incorporating the formate dehydrogenase promoter (pFMD), a signal sequence, and the GCSF gene.
• The expression cassette and zeocin resistance gene were cloned into an E. coli vector.
• The final vector was transformed into Hansenula polymorpha, and GCSF expression was induced using methanol.
• SDS-PAGE and Western blot analysis were employed to verify GCSF expression and identity.

Main Results:

• A functional expression cassette containing the GCSF gene and zeocin resistance marker was successfully constructed and transformed into Hansenula polymorpha.
• Recombinant Hansenula polymorpha colonies resistant to zeocin were selected.
• Induction with methanol resulted in the expression of a protein of approximately 19kDa, confirmed as GCSF by Western blot.

Conclusions:

• The developed expression vector, featuring the pFMD promoter and signal sequence, effectively facilitates GCSF gene expression in Hansenula polymorpha.
• The successful expression of the 19kDa protein demonstrates the capability of this engineered yeast system for heterologous protein production.