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Volume 8
Journal of Biotechnology & Biomaterials
ISSN: 2155-952X
Euro Biotechnology 2018
October 11-12, 2018
October 11-12, 2018 | Moscow, Russia
21
st
European
Biotechnology Congress
conferenceseries
.com
Page 22
Hyper-production and stability of human serum albumin in
Pichia pastoris
through a combination
of medium design and genetic strategies
Saroj Mishra
and
Nitu Maity
Indian Institute of Technology Delhi, India
H
uman serum albumin (HSA) is an important therapeutic recommended for treatment against trauma, burn injury,
hypoproteinemia, hypoalbumenia as well as for maintenance of homeostasis, transportation of hormones and
microelements in blood. In this study, we report medium design and genetic strategies that lead to high production of
this protein in the culture spernatant of
Pichia pastoris
. The codon-optimized gene for HSA was cloned downstream of
α–factor secretory signal sequence and the mature HSA was secreted in the culture supenatant of
P. pastoris
under the
control of alcohol oxidase 1 promoter. Extracellular protein level of 0.12, 0.40, 1.2 g/L were obtained in the un-optimized
medium for 1-copy, 2-copy and 3-copy expression casettes respectively at shake flask level. Factors affecting production
were identified which included initial peptone concentration, methanol concentration and temperature, amongst many
other (pH, aeration, sorbitol concentration, initial inoculum) investigated parameters. A three level factorial design
named central composite design using Plackett Burman response surface methodology was used to optimize the medium
which lead to levels of protein up to 0.075, 0.40 and 0.98 g/L total extracellular protein for 1,2 and 3-copy constructs
respectively. Under these conditions, HSA produced was stable and free of other contaminating proteins in the culture
supernatant. A detailed transcriptome analysis of the recombinant
P. pastoris
, cultivated on unoptimized and optimized
medium lead to identification of several protein coding transcripts which were up-regulated and helped in efficient
HSA production and secretion. These were mapped to biochemical activities linked (and not restricted to) to carbon,
nitrogen metabolism, gene transcription, protein transport and secretion. Additional genetic strategies applied included
modification of signal sequences. Application of optimized medium to these mutants lead to stable production of HSA
with reduced proteolytic degradation of the synthesized protein. This illustrated the robustness of the designed medium
with a production of over 2 g/L protein at shakeflask level. An understanding of the underlying mechanisms is likely to
play significant role in use of
Pichia
system for production of heterologous proteins.
Biography
Saroj Mishra completed her PhD from City University of NewYork, USA followed by Post-doctoral research at Institute Pasteur, Paris, France, VTT
Biotechnical Laboratory, Espoo, Finland and University of California, Davis, USA. She has published more than 87 papers in reputed journals and leads a
large group of scientists working in the area of enviromental biotechnology, whole cell biotransformation and recombinant therapeutics.
saroj98@hotmail.comSaroj Mishra et al., J Biotechnol Biomater 2018, Volume 8
DOI: 10.4172/2155-952X-C5-100