The expression of human leukemia inhibitory factor in Pichia pastoris

Abstract

Background: Leukemia inhibitory factor (LIF) is an interleukin 6 class cytokine that inhibits cell differentiation during cell development. LIF is widely used as an important component in stem cell culture medium for cell therapy and tissue engineering applications, to inhibit spontaneous cell differentiation. However, commercially available recombinant LIF proteins are expensive, unstable, and prone to degradation in prolonged tissue culture, which has limited its applications.

Purpose: We aim to develop a lab-scale method, using a yeast cell, namely, P. pastoris, to produce recombinant hLIF (rhLIF), with novel recombinant protein expression techniques.

Methods: We first designed the structure of plasmid and generated the plasmid using the service of an external supplier. The plasmid was amplified in E. coli using a heat shock method. The amplified plasmid was then linearized and transcribed in the P. pastoris. Methanol was used in the medium to induce the expression of rhLIF in P. pastoris. The resultant rhLIF was tested verified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)

Results: The plasmids were successfully amplified in E. coli. The generation of rhLIF in P. pastoris was verified by the SDS-PAGE results. To our knowledge, it is the first study to express rhLIF using lower eukaryotic yeast.

Future direction: In the future studies, we will study the glycosylation sites of rhLIF using liquid chromatography–mass spectrometry (LC–MS). In addition, the function and stability of the rhLIF will be compared with commercial products.