Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris

Abstract

Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH 5.9 (64 mg/L at 0.02 1/h) compared to pH 5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene‐expression differences between Mut+ and MutS strains of Pichia pastoris

“…Although it is well established that high levels of recombinant protein production affect cell growth, it appears that the reverse may also be true, at least when growth is linked to methanol catabolism. This was in fact demonstrated by intentional growth rate limitation using lower cultivation temperature (Jahic, Wallberg, Bollok, Garcia, & Enfors, ), methanol feeding rates (Raschmanová et al, ; Sinha et al, ), or oxygen levels (Khatri & Hoffmann, ), which all led to higher recombinant protein productivities. It is important to emphasise that these reduced growth rates were accompanied by reduced methanol consumption rates.…”

“…Flow cytometry (FC) was selected to follow the induction of expression as a function of cellular fluorescence and to monitor the behaviour of the population in response to methanol (Broger, Odermatt, Huber, & Sonnleitner, ). Indeed, this convenient combination of reporter protein and analytic tool has recently been used (Raschmanová et al, ), including to develop a methanol biosensor (Takeya, Yurimoto, & Sakai, ). A MutS derivative of the Mut+ strain GS115 was constructed, for the comparison of EGFP production during batch cultivation in bioreactors.…”

Establishment and evaluation of cell and animal models expressing BORIS subfamily 2 variant

“…In our study, we successfully inserted BORIS sf2 into the plasmid pCS-CG, but weak EGFP protein expression resulted in nearly invisible fluorescence in the cells infected by Lenti-pCS-CG C2/A4. A previous study reported cleavage of the fusion protein containing EGFP as a fusion partner and suggested this was probably caused by the presence of substances that degraded the fusion protein (35). Toca-Herrera et al also pointed out that the intensity of EGFP fluorescence emission was related to temperature, pH conditions, and guanidine hydrochloride concentrations (36).…”