Optimization of protein extraction and quantification in the yeast Kluyveromyces lactis

Authors: Alexandra Benčová 1    Tatiana Mančušková 2    Marina Ožbolt 3    Edi Zucca 3   
1 Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia    2 Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia    3 Department of Biotechnology, University of Rijeka, Rijeka, Croatia   
Year: 2017
Section: Biotechnology and Food Technology
Abstract No.: 1632
ISBN: 978-80-972360-1-4

Introduction: Kluyveromyces lactis is a yeast used in biotechnology for production of enzymes. It is simple organism (no WGD compared to Sacharomyces cerevisiae) with lower preference for fermentative carbon metabolism (respiratory eukaryote model), high oxidative metabolism and generation of reactive ogyxen species. It is highly suitable for comparative and evolutionary studies [1,2]. Protein extraction and determination of protein concentration are the base of every proteomic study. Since various approaches are applied in practice, we decided to compare the most commonly used methods in our work. The first part of the work deals with comparison of extraction methods using lytic enzymes, acetic acid and SDS. The second part of the work is focused on comparison of Lowry, Bradford, biuret and absorbance protein assays.

Materials and methods: Yeast cell wall is rigid and the absolute quantification of intracellular protein levels is technically demanding. In practice, various methods are used to break the wall: glass beads for mechanical disruption, SDS and β-mercaptoetanol for chemical disruption, alkaline conditions (NaOH) for cell wall weakening. In this work, we compared procedures for protein extraction from Kluyveromyces lactis according to Jefferson et al. [3], Gbelska et al. [4], Zhang et al. [5] and Haar [6]. Biuret method, Lowry’s method, Bradford’s method, and absorbance at 280 nm [7] were used for protein quantification.

Results: 1-dimensional SDS-PAGE vertical gel electrophoresis was performed to evaluate effectivity and suitability of every tested method. Results showed that enzymatic method is suitable when native conformation of proteins is needed. The NaOH/AA method can be used for the fastest procession and for post-procession by 2D electrophoresis the NaOH/SDS method is suitable. In comparision to the other methods of determination of protein concentration, biuret method lacks sensitivity and it is not suitable for samples with low protein content. Lowry‘s and Bradford‘s methods underestimated protein concentration in K. lactis extract but BSA standard‘s band reached the optimal intensity. A280 method is strongly dependent on purity of protein solution.

Conslusion: 1D profiles of proteins extracted by different methods are similar and the choice depends on every scientist and the aim of his work. Lowry’s and Bradford’s method were shown to be most applicable in practice.

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