Expression and secretion of recombinant ZZ–EGFP fusion protein by the methylotrophic yeast Pichia pastoris

Tang, Jin‐Bao; Zhu, Peng; Yang, Hong-Ming; Sun, Lir-Fang; Song, Shuliang; Ji, Aiguo

doi:10.1007/s10529-008-9714-5

Cited by 7 publications

(3 citation statements)

References 15 publications

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“…Control experiments such as SDS-PAGE, Western blot, or other immunochemical analysis proved that the integrity of the fusion protein is generally of great importance when using EGFP as a fusion tag. Using these additional analyses, some other authors observed no cleavage of a GFP fusion protein (Surribas et al 2006(Surribas et al , 2007Tang et al 2008;Sjöblom et al 2012), indicating that the cleavage we observed is likely to be product-specific.…”

Section: Egfp Was Cleaved From Porcine Trypsinogenmentioning

confidence: 45%

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

et al. 2018

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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.

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Section: Egfp Was Cleaved From Porcine Trypsinogenmentioning

confidence: 45%

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

et al. 2018

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“…In this respect, eGFP fusion proteins may be used with caution when investigating secretory processes in P. pastoris . However, these effects may also be protein dependent, as there were cases reported where GFP was successfully used to evaluate signal sequences [ 59 , 60 ] and some GFP fusion proteins were sufficiently secreted [ 61 , 62 ].…”

Section: Resultsmentioning

confidence: 99%

Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

et al. 2015

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BackgroundTagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar).ResultsHere we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in frame into all 40 plasmids using blunt end or TA ligations, requiring only verification of the orientation. We have established a RSFC vector family for the commonly used protein expression host Pichia pastoris and demonstrated the system with the secretory expression of horseradish peroxidase (HRP). HRP fusions to four tags (Myc, FLAG, His, Strep) and two fusion proteins (GFP and MBP) showed a 31-fold difference in volumetric activities. C-terminal tagging caused in some cases almost a complete loss of function, whereas N-terminal tags showed moderate differences.ConclusionsThe RSFC vectors provide an unprecedented toolbox for expression optimization in P. pastoris. The results obtained with HRP underline the importance of comparing different tags to maximize activities of fusion proteins. In a similar fashion the RSFC strategy can be applied in other expression hosts to screen for optimal promoters, signal sequences or to facilitate the evaluation of (iso-) enzyme families.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0293-6) contains supplementary material, which is available to authorized users.

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“…Many IG-binding proteins have been widely studied and used in the fields of immunology and biotechnology (Dahlbom et al 2008), the most well known examples are staphylococcal protein A (SPA) and ZZ domains (two synthetic IgG-Fc-fragment-binding domains based on the sequence of the domain B of SPA) (Moks et al 1986;Ståhl and Nygren 1997). So far, SPA and ZZ domains fused with GFP or its variants have been developed in Escherichia coli or yeast for recombinant immunoconjugates (Aoki et al 1999;Fujino et al 2002;Huang et al 2006;Tang et al 2008). However, their applications mostly focus on Western blotting.…”

Section: Introductionmentioning

confidence: 99%

Preparation of a bio-immunoreagent between ZZ affibody and enhanced green fluorescent protein for immunofluorescence applications

Yang

Chen

Gao

et al. 2011

World J Microbiol Biotechnol

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In the present study, we constructed plasmid pUC-ZZ-EGFP to express Pro-ZZ-EGFP using ZZ peptide (a synthetic artificial IgG-Fc-fragment-binding protein derived from the B domain of staphylococcal protein A) and enhanced green fluorescent protein (EGFP). Without induction with isopropyl-β-D: -thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.6 × 10(8) M(-1) obtained by competitive enzyme-linked immunosorbent assay, indicating that the ZZ peptide retains the native structure in Pro-ZZ-EGFP. The application of immunofluorescence assay for detecting the Mycoplasma pneumoniae IgG antibody, Pro-ZZ-EGFP, exhibited a good signal comparable in brightness and fluorescence pattern with the signal generated using the fluorescein isothiocyanate-labeled anti-human IgG. The result indicates that Pro-ZZ-EGFP possesses great potential for clinical immunofluorescence IgG test as an alternative versatile fluorescent antibody.

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Expression and secretion of recombinant ZZ–EGFP fusion protein by the methylotrophic yeast Pichia pastoris

Cited by 7 publications

References 15 publications

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

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

Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

Preparation of a bio-immunoreagent between ZZ affibody and enhanced green fluorescent protein for immunofluorescence applications

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