Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris
نویسندگان
چکیده
BACKGROUND Together with the development of optical sensors, fluorometry is becoming an increasingly attractive tool for the monitoring of cultivation processes. In this context, the green fluorescence protein (GFP) has been proposed as a molecular reporter when fused to target proteins to study their subcellular localization or secretion behaviour. The present work evaluates the use of the GFP fusion partner for monitoring extracellular production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris by means of 2D-fluorimetric techniques RESULTS In this study, the GFP-ROL fusion protein was successfully produced as a secreted fusion form in P. pastoris batch cultivations. Furthermore, both the fusion enzyme and the fluorescent protein (GFP S65T mutant) retained their biological activity. However, when multiwavelength spectrofluorometry was used for extracellular fusion protein monitoring, riboflavin appeared as a major interfering component with GFP signal. Only when riboflavin was removed by ultrafiltration from cultivation supernatants, GFP fluorescence signal linearly correlated to lipase activity CONCLUSION P. pastoris appears to secrete/excrete significant amounts of riboflavin to the culture medium. When attempting to monitor extracellular protein production in P. pastoris using GFP fusions combined with multiwavelength spectrofluorimetric techniques, riboflavin may interfere with GFP fluorescence signal, thus limiting the application of some GFP variants for on-line extracellular recombinant protein quantification and monitoring purposes.
منابع مشابه
Expression of the VP2 gene of classical D78 infectious bursal disease virus in the methylotrophic yeast Pichia pastoris as a secretory protein
Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease, an infectious disease of global economic importance in poultry. The expression of heterologous proteins in P.pastoris is fast, simple and inexpensive. In this study, VP2 encoding gene of classical D78 IBDV was amplified using reverse transcription (RT) polymerase chain reaction (PCR) and cloned into pPICZαA vector...
متن کاملP-65: Effective Parameters on the Bovine Follicle Stimulating Hormone Expression in The Pichia Pastoris System
Background: Bovine follicle-stimulating hormone (bFSH) is a heterodimer hormone that consists of a common -subunit which noncovalently associated with the hormone-specific -subunit. During the past 15 years, the methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production because it is able to use methanol as a sole carbon and energy source. Th...
متن کاملCloning of fusion (F) protein gene of peste des petits ruminants virus (PPRV) in secretory Pichia pastoris vector
With advent and development of DNA recombinant technology and advantages of p. pastoris expression system, fusion (F) protein of PPRV expression, because of effective immunodominant role could be an appropriate candidate for production of recombinant vaccine against PPR disease. In this study, F gene of PPRV Nigeria 75/1 strain (1637 bp) was amplified using RT-PCR and purified. It was then cl...
متن کاملIn silico design and expression of a novel fusion protein of HBHA and high antigenic region of FAP-P of Mycobacterium avium subsp. paratuberculosis in Pichia pastoris
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants and there has been a shift in the public health approach to MAP and human diseases like Crohn's disease. The prevention of infection by MAP in ruminants is thought to deter the high impact of economic losses in the level of dairy industry and possible spreading of this pathogen in dairy prod...
متن کاملExpression of recombinant GFP-actin fusion protein in the methylotrophic yeast Pichia pastoris.
The integrative vector pPIC3 for the yeast Pichia pastoris and a cDNA fragment encoding a fusion protein consisting of green fluorescent protein (GFP) and actin 5C of the fruit fly Drosophila melanogaster were used to construct a pPIC3-GFP-actin 5C expression plasmid. The P. pastoris host strain GS115 was transformed with the pPIC3-GFP-actin 5C carrying HIS4 as a selective marker. The transform...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Microbial Cell Factories
دوره 6 شماره
صفحات -
تاریخ انتشار 2007