Alternative approach for recombinant protein expression in the mammary gland

Expression of active fluorophore proteins in the milk of transgenic pigs bypassing the secretory pathway

9.3 Alternative approach for recombinant protein expression in the mammary gland

Mammary gland of transgenic animals as a bioreactor has been extensively exploited to produce recombinant proteins at large scale. In case of human lactoferrin, a yield of grams per litre has been achieved (van Berkel et al., 2002;

Yang et al., 2008; Goldman et al., 2012), but in some experiments only minute amounts of recombinant protein were obtained (Platenburg et al., 1994;

Niemann et al., 1999; Tong et al., 2011). To achieve high expression of recombinant proteins in the milk of transgenic animals, a secretory signal sequence along with a mammary gland-specific promoter element, like those of the casein, lacto-globulin and lacto-albumin genes are commonly used (Whitelaw et al., 2016).

Here, I describe an alternative approach to produce recombinant proteins in the mammary gland. In this study, reporter transposon pig lines carrying the Venus, or the mCherry constructs (see 9.2) were exploited. The reporter constructs were driven by the ubiquitously active CAGGS promoter, and devoid of a signal peptide sequence, resulting in robust expression and a cytoplasmic localisation of Venus and mCherry fluorophores (Mukherjee et al., submitted).

However, high levels of Venus and mCherry proteins were readily detected in sow milk samples from the two lines, respectively. For the Venus fluorophore an expression level of 0.27 – 0.38 g/l, and for mCherry an expression level of 0.2 – 0.25 g/l were determined. Considering that porcine milk contains 6–8 g of protein

per litre (Klobasa et al., 1987), this equals to 2.5 – 6.3 % of the total protein content (Mukherjee et al., submitted).

The cumulative data suggested that the recombinant Venus and mCherry are not transported via the secretory pathway, but that exfoliated cells of the udder epithelium carried Venus or mCherry into the milk. During lactation, the udder epithelium represents one of the most highly proliferative tissues in mammals (Chepko and Smith, 1997), and undergoes substantial remodelling (Strange et al., 1992). The somatic cell count of porcine milk is higher than in other mammals, and has been determined between 10⁸ to 10¹⁰ cells per litre (Magnusson et al., 1991; Scharek-Tedin et al., 2015). The milk cells represent apoptotic cells, but also vital lactocytes (milk secretory cells), epithelial and immune cells (Hassiotou and Hartmann, 2014). The colostrum typically contains a relatively high number of immune cells, which declines in mature milk. In healthy sows after the first week of lactation, the vast majority (>95%) of milk cells consist of lactocytes and mammary myoepithelial cells (Scharek-Tedin et al., 2015).

The expression analysis of blood fractions suggested minute or un-detectable levels of Venus and mCherry fluorophore in the blood serum.

Here, in this study an alternative approach of harvesting recombinant proteins in large scale through somatic cell fraction of milk has been described.

The data suggest that SB transposon-mediated gene delivery prefers transcriptionally permissive genomic loci. For the development of SB transposon based DNA vaccines, as well as the SB transposon based recombinant protein production, the targeting of transcriptionally permissive loci in the porcine genome is an important advantage. Thus the here established methods and findings provide the basis for more advanced approaches.

Acknowledgements

Being Doctor of Philosophy (PhD) is undoubtedly a great achievement. When I revisit last three year of my PhD research stay in Germany, I evaluate my achievements in regards of some other parameters, which provided me some extra satisfaction. Commencing the journey from a phase of lowest position of sinusoidal curve of my academic career, my PhD research stay repositioned me at the peak. These three years of most productive and intensive stage of my life was incubated with an excellent environment with dynamic minds, including both from the scientific community and outside of the academic environment. I am grateful to all those seen and unseen hands which pushed me forward towards the right path of knowledge, experience and enlightenment.

Leader is a person, who gives credits of an achievement, to its team members, always being behind the curtain. On this auspicious occasion, I would like to unveil that person to extend my sincere thanks to my ‘Guru’ in true sense, Dr. Wilfried August Kues, for his able guidance, appreciation, suggestions, constructive criticism, providing me knowledge to develop concepts, indefatigable encouragement from the inception of this project, giving critical comments wherever and whenever required, very worthy discussions, being counsellor and morale booster and providing fatherly affection and care during this period.

I offer a special thanks to Prof. Dr. Pablo Steinberg and Prof. Dr. Klaus Osterrieder, for their careful and professional guidance and constant encouragement. They have always been kind and helping. They always monitored the progress of my work and were available when I needed their advice.

I am also thankful to Prof. Dr. Heiner Niemann, Director, Institute of Farm Animal Genetics, FLI, Mariensee, for providing me the necessary research and academic facilities required for my study and research work.

I would like to convey my sincere gratitude to Indian Council of Agricultural Research (ICAR) for the financial support throughout my research stay and constant monitoring of the progress of my research.

Shiddhartha Da, Sujoy Da, Aishik and Debasmita – as I don’t have any word to express my gratitude for their critical supports, I just want to say ‘thank you’ for their affection and truthfulness and moral support during all the lows of my life.

I would also like to express my gratitude for sincere efforts of Maren, Sabine, Doris, Patrik, Karin and Tony for their help, patience, support and valuable suggestions with all their expertise to make my research endeavour to a success.

Without friends life would have been a monochromic canvas. I am feeling privileged to thanks all those wavelengths that illuminated my canvas with rainbow. I am truly thankful to the almighty for having friends like Roberto, Sandra, Rao sir, Dharmendra sir, Rolf, Diego. I specifically feel blessed to have friend like Roberto. I just want to say “Thank you”, for all those adventures. I am equally thankful to Dorothee, Amanda, Jun, Ilka, Ronja …….. for their constant efforts to make my life happening.

It will be injustice if I don’t express my thanks to all those public bicycles which have taken all the pains to carry me to the every corner of the nearby swamp areas.

My life has been always embellished with the blessing, love and uninterrupted support from my parents Mr. Bijan Mukherjee, Mrs. Dhira Mukherjee and my sis Jayanti Mukherjee whose sustained encouragement and love always pushed me to achieve new heights. I also owe a lot to facebook, youtube, Whats App, Gmail and skype. It is their presence which brought my two

worlds together and it is their presence that didn’t allow me to feel the distance.

As there are not enough words to express my profound feeling of reverence and love for my family, I just wanted to say, I owe a lot to them.

And many others who are not mentioned, but no one is forgotten……

Finally, as though I am very small before him, still I would wish to acknowledge ‘God’ without whose blessing this small piece of work would have never been successful all through the way of truth and love and also creating such a beautiful world for us with all amenities.

Thank you all………

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