Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

doi:10.1016/0264-410X(91)90011-T

Vaccine

Volume 9, Issue 12, December 1991, Pages 901-906

https://doi.org/10.1016/0264-410X(91)90011-T Get rights and content

Abstract

Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded > 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.

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Cited by (87)

Quantitative microcapillary electrophoresis immunoassay (mCE IA) for end-to-end analysis of pertactin within in-process samples and Quadracel® vaccine
2021, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
Pertactin (PRN or P69) is a cell-surface adhesion protein of B. pertussis and is one of the components of the aP vaccine [14]. Since its discovery, PRN recovery from cell cultures is low in comparison to other antigens [15–17]. Hence, it is important to closely monitor each production step for PRN yield, to ensure maximum recovery.
Protein concentration is an important attribute in the production of subunit or component-based vaccine antigens. Rigorous monitoring of protein concentration is required to identify potential areas for yield improvement. The current GMP method for quantitation is the plate-based ELISA which requires numerous hands-on steps and has low sensitivity in comparison to new microfluidic systems. To address this issue, a sensitive automated microCapillary Electrophoresis ImmunoAssay (mCE IA) method was developed to accurately separate and quantitate pertactin (PRN), an important antigen of the modern acellular Pertussis (aP) vaccine. PRN is reported to be a low-yielding antigen; thus, it is critical to observe its concentration throughout its manufacturing process. First, a primary antibody for PRN was identified to establish suitable immunoprobing conditions for detection of PRN over a wide linear dynamic range that spans 3 orders of magnitude. Next, the pre-adsorbed PRN Drug Substance (DS) was used as a reference standard to quantitate PRN samples against a calibration curve with adequate accuracy and precision. Four representative samples including three in-process steps and final adjuvanted drug product: Quadracel®, were examined to demonstrate the capability of mCE IA to quantitate PRN with high sensitivity and specificity. The matrices of the selected samples contain additional components (e.g. other proteins, growth factors, cell culture media, residual ammonium sulfate, and aluminum adjuvant) often making the quantitation of PRN challenging. The specificity and method linearity were demonstrated by spiking pre-adsorbed PRN DS into the four representative samples. In addition, it was shown that reportable concentrations of PRN for nine downstream process steps as analyzed by our method is comparable to concentrations obtained with ELISA. Most importantly, this study demonstrated that our method’s quantitative accuracy is independent of matrix components, as each sample undergoes extensive dilution. This allows for seamless end-to-end analysis of PRN from fermenter harvest, through to complex downstream process samples to adjuvanted drug products. Finally, for the first time the developed and qualified mCE IA method was shown to quantify PRN throughout the entire manufacturing process to provide rapid feedback for process optimizations allowing for accurate yield and step-loss calculations.
Prevention of aggregate formation through mechanism analysis in refolding of recombinant pertactin from Escherichia coli
2017, Process Biochemistry
Production of natural pertactin for pharmaceutical use is limited by its low abundance. In this study, recombinant pertactin was highly expressed in the form of inclusion bodies in E. coli. However, up to 75% of the soluble turned out as aggregates when refolding by pulse-fed batch dilution. The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein, resulting in aggregation between proteins with different folding states. The key factor for prevention of aggregate formation was to improve the synchronization of refolding. For this purpose, flash-batch dilution was conducted at a scale of 5 L and achieved a monomeric refolding yield of above 70%. Aggregates formed were efficiently removed along with impurities by one-step chromatography of Ni-resin. The purity of monomeric pertactin was >98%. An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%. The purified protein was characterized by MALDI-TOF, circular dichroism, fluorescence, HPLC and DSC, and showed similar physiochemical properties compared to its natural counterpart. Animal study showed similar immunological responses and antibodies elicited demonstrated a comparable reactivity.
Development of Process to Produce Recombinant Component for Acellular Pertussis Vaccine
2017, Current Developments in Biotechnology and Bioengineering: Human and Animal Health Applications
This chapter describes new technologies related to the production of acellular pertussis vaccine (DTaP). Although many attempts have been made in past decades, pertussis, also known as whooping cough, remains endemic and the number of cases has increased. Thus new ways are needed to prevent this disease, with fewer side effects. Therefore a case report of the development of a process to produce the fraction of immunogenic protein used to produce an acellular vaccine (DTaP) is presented. In this case, recombinant technology is used through which a portion of the gene encoding pertussis toxin is expressed in a host cell and purified, aiming to produce a subunit vaccine.
Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation
2014, Journal of Bioscience and Bioengineering
Lysozyme acts as an important defensive factor in innate immunity due to its well-recognized bacteriolytic activity. Here we describe the production and performance of human lysozyme-like 6 (LYZL6), a novel human c-type lysozyme homolog. A synthetic codon-optimized cDNA encoding the intact amino acid sequence of LYZL6 was cloned and expressed in Pichia pastoris SMD1168. Bioactive LYZL6 was successfully produced as a single major secreted protein with a molecular weight of 15 kDa, and exhibited bacteriolytic activity against Micrococcus lysodeikticus. The expression conditions were optimized, and the highest expression level of LYZL6 occurred when the recombinant strain was induced with 1.5% methanol under pH 4.5 at 24°C for 96 h. When high cell density fermentation of the recombinant P. pastoris was performed using a fed-batch strategy for totally 125 h in a 30 L fermenter, the dry cell weight and the extracellular lysozyme activity were increased to 116.3 g/L and 2340 U/mL, respectively. The LYZL6 protein concentration was 331 mg/L of fermentation supernatant, and the specific activity of LYZL6 towards M. lysodeikticus was 7069 U/mg. Therefore, we proved that LYZL6 is an antibacterial protein, suggesting a potential application of LYZL6 as an antimicrobial agent, and Pichia expression system for LYZL6 was successful and industrially promising.
Expression of Apostichopus japonicus lysozyme in the methylotrophic yeast Pichia pastoris
2011, Protein Expression and Purification
Apostichopus japonicus (sea cucumber) is one of the economically important farmed echinoderm species in Northern China. As a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, a lysozyme gene from A. japonicus was cloned by PCR and expressed in Pichia pastoris using the expression vector pPIC9K. The expressed lysozyme had a molecular mass of ∼14 kD, as shown by SDS–PAGE and Western-blotting. The expression condition was optimized, and the highest expression level was achieved by induction with 1% methanol at pH 5.0 for 120 h. The recombinant lysozyme was purified by affinity chromatography using a Ni–NTA column. The specific activity of the purified lysozyme was 34,000 U/mg using Micrococcus lysodeikticus as substrates. It exhibited antimicrobial activity toward M. lysodeikticus, as detected by growth inhibition on agar plate and turbidity assay, suggesting a potential application of A. japonicus lysozyme as an antimicrobial agent in A. japonicus aquaculture.
Comparison of recombinant and native pertactin of Bordetella pertussis
2011, Vaccine
Citation Excerpt :
Thanks to the recombinant DNA technology, some studies have been tried to obtain expression of recombinant Prn (r-Prn) in Escherichia coli, Baculovirus insect cell and Yeast expression systems [15–19]. So far, most of these studies have focused on the immunogenicity, protection efficacy or the antigenic epitope(s) of recombinant protein [16–20]. However, physical and biochemical characterisation of r-Prn have not been investigated systematically.
Bordetella pertussis pertactin (Prn) is an important attachment factor and protective immunogen, which serves as a component in most acellular pertussis vaccines (APVs). Here, we over-expressed recombinant Prn (r-Prn) without an affinity tag using an Escherichia coli expression system. Compared to the native Prn (n-Prn) from B. pertussis, the recombinant protein showed a comparable reactivity in Western blotting and ELISA as well as a similar structure as analyzed by circular dichroism, fluorescence spectroscopy and size-exclusion chromatography. Furthermore, parenteral immunization of mice with native or r-Prn produced a similar increase in serum anti-Prn antibodies and similar protective activity following aerosol challenge. Our results indicate that the recombinant protein approach may be useful for developing a potential component of APVs as well as an antigen material which can be used in both clinical epidemiological evaluation and laboratory vaccine evaluation studies. Moreover, these studies also provide further evidence for the role of Prn in pertussis immunity.

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PaperRecombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties

Abstract

Vaccine

Lancet

Anal. Biochem.

Progress report on pertussis immunisation

Am. J. Public Health

WHO Report of expert committee on biological standardisation

WHO Technical Report Series

Pertussis immunisation and serious acute neurological illness in children

Br. Med. J.

Reactions to whole-cell pertussis vaccine

Mutants of pertussis toxin suitable for vaccine development

Science

Placebo-controlled trial of two acellular pertussis vaccines in Sweden — protective efficacy and adverse effects

Lancet

Pathogenesis in Bordetella species

J. Infect. Dis.

Identification of a 68-kilodalton outer membrane protein as the major protective antigen of Bordetella bronchiseptica by using specific-pathogen-free piglets

Infect. Immun.

Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells

Characterisation of the protective capacity and immunogenicity of the 69-kD outer membrane protein of Bordetella pertussis

J. Exp. Med.

Dissecting human T-cell responses against Bordetella species

J. Exp. Med.

Paper
Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties