Inan Lab on Protein Engineering

OVERVIEW

Many human diseases are related to the absence or malfunction of a protein. Functional proteins need to be produced ex vivo and administered to patients to treat the disorders. In recent years, the number of recombinant proteins used for therapeutic applications and biochemical characterization has increased dramatically. In some applications relatively small quantities of recombinant proteins are needed, whereas in other cases, such as therapeutic applications, almost metric quantities are needed. A range of heterologous expression systems has been developed to supply the demand for recombinant proteins. Among these systems, yeast offers certain advantages over prokaryotic hosts which include an intracellular environment that is more suitable for the correct folding of proteins.

Pichia pastoris (Komagatella phaffii) developed as a single cell protein in 70’s and evolved into a promising expression host for the production of recombinant proteins. It has successfully been used in the production of more than 1000 recombinant proteins with almost half biopharmaceuticals along the way. Human serum albümin, a vaccine against Botulism neurotoxin, hormones like insülin, human proteases like ocriplasmin, and protease inhibitors like ecallantine are some examples, while the last two have been approved by the FDA.

P. pastoris exhibits fast growth to high biomass concentrations (more than 150 g dry cell weight per liter) in bioreactors on inexpensive media like bacteria. And also, like mammalian cell culture, it has an ability to perform typical eukaryotic post-translational modifications such as proteolytic processing, disulfide bridge formation, and glycosylation. Genetic manipulation of P. pastoris is easily achievable as well leading to remarkable efforts for the reengineering the P. pastoris for biopharmaceutical production. At the current stage, P. pastoris has successfully glycoengineered to produce humanized glycoforms of human proteins. Having high secretion efficiency with a low amount of endogenous proteins is another key feature of the Pichia system.

RESEARCH INTEREST

Strain Development

Our research mainly focuses on understanding and improving the Pichia expression system to provide the P. pastoris user community with improved system components and methods. The basis for our efforts is trying to illuminate the regulation mechanisms of commonly used promoters to enhance their capacity and also to identify new regulatory sequences which may represent a potential alternative to routinely used promoters. For example, ADH genes which involve in P. pastoris ethanol metabolism were identified and characterized recently in our previous studies. The gene responsible for the consumption of ethanol was determined as the ADH3 gene and it was highly expressed on ethanol as substrate source. However, the regulation of the ADH3 promoter at the molecular level is not known yet. The elucidation of the regulation of the ADH3 promoter will enhance its utilization for recombinant protein production and will enable the design of stronger promoters for higher protein production at a large scale.

Process Development

The production yields of heterologous proteins are not only influenced by host strain but also the culture conditions. Therefore, in order to develop a high-yield bioprocess, different strategies should be applied including both genetic and process engineering approaches. Since recombinant protein production kinetics are highly dependent on protein-specific factors, it is not possible to end up with a single optimal strategy for the process. Our work covers both strain and process development. We aim first to create a high yielding production strain for the protein to be produced and then develop a bioprocess concerning the technical possibilities and limitations of industrial scale applications.

Selected Publications

Karaoglan M, Erden Karaoglan F. and Inan M.. Functional analysis of alcohol dehydrogenase (ADH) genes in Pichia pastoris. Biotechnol Lett. 2016 Mar ; 38 (3): 463-9. doi:10.1007/s10529-015-1993-z.

Karaoglan M, Karaoglan FE, Inan M. Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris. Protein Expr Purif. 2016 May ; 121 112-7. doi:10.1016/j.pep.2016.01.017.

Khasa YP, Conrad S, Sengul M, Plautz S, Meagher MM, Inan M. Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion. Yeast. 2011 Mar ; 28 (3): 213-26. doi:10.1002/yea.1832.

Jacobs PP, Inan M, Festjens N, Haustraete J, Van Hecke A, Contreras R, Meagher MM, Callewaert N. Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate. Microb Cell Fact. 2010 Nov ; 9 93. doi:10.1186/1475-2859-9-93.

Kane J, Inan M and Saraf R.F. Self-Assembled Nanoparticle Necklaces Network Showing Single-Electron Switching at Room Temperature and Biogating Current by Living Microorganisms. ACS Nano. 2010 ; 4 (1): 317-323. doi:10.1021/nn901161w.

Contact

Inan Lab on Protein Engineering

Group Leader

Mehmet İNAN
mehmet.inan@ibg.edu.tr
+90 232 299 41 00 (5271)
+9 02322994177