Survey of intracellular proteins extraction methods from pichia pastoris
Yuli Wang, Yang Lei, Yangyong Lv,Le Wang,Hongwei Wei,YuansenHu
(Collegeof Biologicalengineering, Henan University of Technology, Henan Zhengzhou, 450001.)
Abstract:In order to explore a stable and effective extraction method of intracellular proteinsfromPichia pastoris, the broken efficiency of cell walland the release amount ofintracellular proteinunderdifferentcell breaking conditionswere investigated and compared in this paper. The results showed that broken efficiency of hot alkali combined with high-pressure homogenizing method was higher than that of enzyme hydrolysis,hot alkali treatment and high-pressure homogenation,respectively.Suspended medium had little effect on the broken efficiency of yeast cell, but hadsignificanteffect on theprotein release yield.The results indicated that optimal condition for intracellular proteins extractionwas 30%(wet weight, w/v) of yeast cells suspend in 50mM phosphate buffer(pH10.0) , water bathed at 60℃for 2hours,homogenized twiceat 100MPa pressure.The broken efficiency of Pichiapastoris cell couldreach 87.6% and the protein yieldwas 35.48gper100gcells.
Keywords:Pichia pastoris, high temperature and alkali treatment, high pressure homogeniza- tion, cell wall breaking, protein extraction.
1 Introduction
The methylotrophic yeast Pichia pastoris has become one of themost widely used expression systems for recombinant proteinproduction(Jia-kunDaietal., 2012). More than 500 kinds of proteins have been successfully expressed since the beginning of 1980s;Part of the productshasbeen successfully used inaquatic breed,feedingprocessing and other industries(Rui-juan Lou etal., 2010).With the increasing application of this genetic engineering yeast in fermentation industry,a large number of waste yeast residualare produced every year.At present,the yeast mud is mainly used as fertilizer matrixafter dry compost, which reducesthe added value it should have.
Pichia pastoris contains about 50% proteins, 4%-7%ribonucleic acid (RNA),
more than 20% polysaccharideand other nutrients;extraction of these intracellular
materials from yeast cells is significant for the maximum utilization of yeast resource.
However,due to the thick cell walland firmnetwork structurecomposed of dextrin,
mannose and protein(Cui-zhu Yang etal.,2006), extraction of intracellular proteinsis
faced with many restrictions.So far,yeast cell breaking methods have been reported
in many literatures, such as ultrasonic method,hot acid treatment,biological enzymatictreatment(Yan Li,2008; Ding-wei Cui, 2010;Su-qingZhang,2008).Butthere arestillmany drawbacks: ultrasonic method produces high temperatureto makeproteindenaturation(Jun-huaZengetal., 2005)enzyme hydrolysismethod is costlyandlead to the product inhibition(Bin Wang etal.,2013).In this studysuitablemethodsof cell wall breaking andthe extractionof intracellular proteinsfrom Pichiapastoris were investigated, and the optimal breaking condition was obtained.
2 Materials and Methods
2.1 Experimental materials
Freshpichia pastoris mud (a fermentation corporation,Luoyang Henan).Yeast lyric enzyme(Pang Bo Biological Engineering Limited Company,Nanning Guangxi).
2.2 Equipment
NS1001LHigh pressure homogenizer(Italy Nrio GEA Company);Delta 320Acidity meter(Mettle Toledo Company).TDL-5ACentrifuge(Shanghai Fichal Analysis Instrument Co., Ltd.); DK-S24 Thermostat water bath (Shanghai Jing Hong Experimental Equipment Co., Ltd.);SHA-B Thermostatic oscillator(Jintan science and Technology Instrument Co., Ltd.).
2.3 methods
2.3.1pichia pastorismud pretreatment
Freshpichia pastoris mud was washed by distilled water(2volumesofyeast
mud)and centrifugated and precipitationwas collected, thenwashed again by
distilledwater (1 volume ofyeast mud)toremove the salton the surface of Pichia
Pastoriscells.Thendistilled waterwas added to the pichia pastoris cellmud to make
theconcentration of cell suspension 30% (w/v) andthe moisture content of the pichia
pastoris cells mud was determined( GB5009.3-2010).
.2.3.2Biological enzymatic hydrolysistreatment
500mLpichia pastoris cellsuspension wasequally divided into 5 parts,then 0.1‰，0.2‰，0.3‰，0.4‰and 0.5‰（w/v）of yeast lyric enzymewere added to thepichia pastoris cellsuspensionrespectively for 12 hours at 50℃.After enzymatic hydrolysis,cell breaking efficiency was investigated; supernatant was collected and protein yield was measured.
2.3.3Alkali-heatingtreatment
500mL pichia pastoris cellsuspension wasequally divided into 5 parts,then adjustedpH to 7, 8, 9, 10, 11respectively, water bath for 2 hours at 60℃,stirred every 10min. After alkali-heating treatment,cell breaking efficiency was investigated; supernatant was collected and protein yield was determined.2.3.4High-pressure homogenizing treatment
AdjustpH of 1000mL cellsuspension ofpichia pastoristo 8.0, divided equally into 5 parts.Eachpartwascarried out with high-pressure homogenizing,the homogenized pressure were 40MPa, 60MPa, 80MPa, 100MPa and 120MPa, respectively. After homogenization treatment,cell breaking efficiency was investigated; supernatant was collected and protein yield was determined.
2.4.5Effect of breaking timesonprotein yield ofpichia pastoris cells
Distilled water,50mM phosphate buffer (pH8.0),0.15mol/LNaCl were used to maketheconcentration ofpichia pastoriscell suspension30%.The homogenizing pressurewas 80MPa; thehomogenizing number was 1 to 5 timesrespectively.
2.4.6Alkali-heating and high-pressure homogenizingtreatment
Adjusted pH of each 1000mLpichia pastorissuspensionto 7.0 and 8.0 respectively,and then water bath for 2 hours at 60℃.After the bath, the two samples were equallydivided into four parts,homogenized under 60MPa, 80MPa, 100MPa and 120MPa conditions respectively. After homogenization treatment,cell breaking efficiency was investigated; supernatant was collected and protein yield was measured.
2.3.7Effect of cell concentration on protein yield
50mM phosphate buffer (pH8.0)was used to makeconcentration ofpichia pastorissuspension10%，20%，30%，40% and 50%（w/v, wet weight）with each volume of 200ml.Water bath for 2 hours at 60℃,homogenized twiceunder pressure of 100MPa.After homogenization,supernatant was collected and protein yield was measured.
2.3.8Determination of breaking efficiency ofpichia pastoriscells
1mL of pichia pastorissuspension before and after treatmentwas selected and observed usinghemocytometerafter dilution.The number of yeast with intact morphology was counted.Each sample was counted 5 times, and the average value was obtained.The numberdifference between intact cells before (Nb) and after (Na) treatment was got, which was designated Nb-a; cell breaking rate (%) was the ratio of Nb-a /Nb.
2.3.9Yield determination of released protein
The cell debriswasremoved after centrifugation, supernatant was collected and protein concentration was measured using Bradford method after dilution.The released protein yield was the ratio ofproteins in supernatant and dry weight ofpichia pastorismud.
3.Results discussions
3.1 Effect of Biological enzymatic on cell wall-breaking
Yeast cell wall breaking enzyme is composed ofβ-glucanase, mannose xylanase and chitinase, which can degrade cell wall polysaccharide, release the intracellular proteins.The results showed that with increased amount of wall breaking enzyme, theefficiency of cell breakingwashigher (Fig.1).When thedosage of enzyme was between 0.1‰-0.4‰, the efficiency of cell breaking changed significantly, and yield of released protein also increasedextremely.When the dosage of enzymewas morethan 0.4‰,increased protein yieldwas not obvious.In conclusion, the effect of single biological enzymatic treatmenton yeast cell wall-breaking waslimited. The results indicated that maximum efficiency of cell breaking was about 20%, and the releasedprotein yield was also lower.
Fig.1Effect of Biological enzymatic treatment on cell wall-breaking
3.2 Effect of alkali-heating on cell wall-breaking
Fig.2Effect of alkali-heating treatment on cell wall-breaking
Alkali-heatingtreatment showed that when thepH wasbelow 8.0,the broken efficiencywaslow;with the increase of pH, the cell breaking efficiency improvedsignificantly.Cell breaking efficiency couldreach 36.2% when pH was 11.0.The change trend of protein release yieldwas consistent with the trend of cell breaking efficiency.The results also indicated thatextending the heat treatment time or increasing alkalinitycould make the color of the protein solution deeper with a special smell of sulphide. Thisphenomenon mightbe due to protein denaturationcaused by the reaction between breakage of disulfide bond of the released intracellular proteins and alkali.
3.3 Effect of high-pressure homogenizingtreatment on cell wall-breaking
The results of the high pressure-homogenizingtreatmenton cell wall-breakingindicated that when the homogenized pressurewas less than 60MPa,thecell breaking efficiency waslow.When the pressure increased to 100MPa,the efficiency of yeast cell breaking changedsignificantly, and protein release yield also increasedaccordingly (Fig.3).The results indicated that appropriate homogeneous conditionshould be considered in the process of production, because higher pressure couldlead to damage of equipment, which waseasily to increase production costs.
Fig. 3 Effect of high-pressure homogenizingtreatment on cell wall-breaking
3.4 Effect ofHomogenizedtimes and suspension medium on cell wall-breaking
Homogenizingtimes had a great influence onthe efficiency of yeast cell breaking in suspension.Results showed that, more than 90%of the yeast cells could be brokenafter 3 timesof homogenization,the intracellular protein releaseyieldalso increased accordingly (table 1).The efficiency of cell breaking and the protein release yield were not significantly improvedwhen homogenized time were more than 3.The results also indicated that higher broken efficiency and protein release yield were obtained when using 50mM phosphate buffer (pH 8.0) and0.15MNaCl as suspended medium than that of distilled water.
Table1. Effect ofHomogenizedtimes and suspension medium on cell wall-breaking (三线表格)
Homogenizing times / Broken efficiency（%） / Protein release yield（g/100g）Distilled
water
water / phosphate buffer solution / NaCL solutionn / Distilled
water / phosphate buffer / NaCL solution
1 / 53.2 / 60.3 / 58.1 / 20.75 / 24.55 / 22.62
2 / 76.6 / 78.5 / 78.8 / 30.07 / 35.40 / 31.13
3 / 90.1 / 92.5 / 90.9 / 35.21 / 37.46 / 36.36
4 / 91.2 / 93.6 / 92.8 / 36.02 / 37.90 / 37.03
5 / 92.5 / 94.1 / 92.6 / 36.90 / 37.64 / 36.88
3.5 Effect ofalkali-heating and high-pressure homogenizing on cell wall-breaking
The effect on the yeast cell breaking efficiencyandprotein yield of two conditions of pH9.0 and pH10.0were studied and compared under condition of water bath for 2 hours at60℃and 100MPa pressure homogenized twice. The results indicated that withincreasing of homogenization pressure,theefficiency of cell breakingand the protein yieldincreased at pH 9.0 and pH10.0.Theefficiency of yeast cell breaking can reach 87.6%whenhomogenizedtwice at pH 10.0,it’s;both efficiency of cell breaking and protein yieldwere higher than that of pH9.0.
Table2.Effect of alkali-heating and high-pressure homogenizing on cell wall-breaking
homogenizing pressure（MPa） / Broken efficiency（%） / Protein release yield（g/100g）
pH9.0 / pH10.0 / pH9.0 / pH10.0
60 / 51.1 / 65.5 / 19.80 / 26.20
80 / 69.4 / 79.1 / 27.41 / 32.29
100
100 / 78.3 / 87.6 / 30.77 / 35.48
120 / 86.6 / 89.1 / 35.07 / 35.64
3.6Effect of cell concentration on the extraction of intracellular proteins
Cell concentration on the extraction of intracellular proteins was also investigated. The results indicated that protein release yielddecreased with the increasing of cell concentration (Fig.4).The cell concentration was less than 30%, the protein release yielddecreasedextremely whilethe downtrend slowed down when theconcentration was higher than 30%.In conclusion, low concentration of cellsled to higher broken efficiency, however, the protein yield was less,andthe production cost of protein extraction was higher compared with that ofhigh densitycells.Appropriatecell concentration was favorable for the subsequent production.
Fig4. Effect of cell concentration on the extraction of intracellular proteins
4.Discussions
In recent years, different methods of yeast cell wall breaking have been developed,but each method has its own limitations(Cui-zhu Yang etal.,2006).The researchers use different methods according to the characteristics of the acquired products.Previous studiespaymore attention to obtaining highercell breakingefficiency,and more products as possible;quality of the products and industrial production costs are ignored.Chen(2014)and other researchersfound that using high pressure cell breaking apparatus could reach highest efficiency.After breaking for 10 times,the protein release yield and protein activity were highest.The scale production of this method could increasethe cost ofproduction;it wassuitable for the extraction of the high value material from cells.Wu(2007)combined high-pressure homogenizing,enzymatic dissolution and temperature variationsto study yeast extraction, the efficiency of protein release could reached 86.5%.In this study,wecombinedenzymatic treatment and high-pressure homogenizingto broke the cell walls, however, the effect was not obviousdue to the its thickeningin the late fermentation stageofPichia pastoris.Li(2008)found thatalkali could lysesyeast cellmodestly and broken efficiency was better compared with salt method in the study of nucleic acid extraction.In this study,combination of alkali-heating and high-pressure homogenizing could lead to higher cell broken efficiency compared with enzymatic, alkali-heating and high-pressure homogenizing respectively.In this paper,in consideration of production cost, use and properties of the extracted proteins,the optimal conditions of the alkali-heating and high-pressure homogenizing method was that pH10.0,water bath for 2 hours at60℃,100MPa pressure homogenized twice,under this conditionthe ideal efficiency of yeast cell breaking couldreach 87.6%.
In this paper, we found that cell suspension medium had little effect on the efficiency ofcellwall broking,but the effect on theprotein yield was significant, which was similar to the researchof Su(1990).Of the three kinds of medium, the highest yield protein was obtained from phosphate buffer solution, followed by sodium chloride solution.This result mightbe related to the solubility of intracellular protein,in dilute salt solution the solubility of protein increased,whilein waterthe proteinusually existed ascolloid state,which was easilycross-linked by cell debris,then cell debriscombined with protein were collected as cell waste in centrifugal separation.
Reference
Bin Wang, Li-na Huang, Li-juan Qiuet al. (2013).Study on moderate wall-breaking preparation lipoprotein of Yeast Source.China Biotechnology,33(12) :92-96.
Cui-zhu Yang, Yan Li, Nan Ruan et al.(2006). Study and applications of technology about breaking yeast cell w all.Food Science and Technology,(7): 138-142.
Ding-wei Cui, Xue-chao Hu,Shan-Shan Bao, Kai Zhang et al. (2010).Research progress in enzymatic disruption ofmicrobial cells.Institute of Microbiology, 37(11): 1672−1678.
Jia-kun Dai, Yan Li, Qi Ma et al. (2012).Optimization of ultrasonic treatment for recovery of recombinant protein from Pichia pastoris.Food Science, 33(10):57-60.
Jun-huaZeng, Chang-lu Wang, Min Zhang et al. (2005).Study on releasing S-Adenosyl-L-M ethionine by breaking yeast cells.Amino Acids& Biotic Resources,27(1):49-51.
National standards of thepeople's Republic of china, Food safety national standards-Determination of moisture in foods GB5009.3-2010
Rui-juan Lou,Li-long Luo, Xia Zhang et al. (2010).Research progress and prospects on Pichia pastoris.Journal of Biology,27(5):73-76.
Run-jiao Wu, Zhen-yang Liu, Xiu-ting Zhang et al. (2007). Extraction of Yeast Monosodium Glutamate from Waste Beer Yeast by the Synthetic Technique of Breaking and Dissolving Cell Wall. Liquor - M aking Science & Technology,(11):95-96.
Su -qing Zhang, Gui-lan Kao et al. (2008).Thestudy of theeffect for the shiver of the yeast cell using acid-heat shiver.Journal of Inner Mongolia Agricultural University, 29(3):15-18.
Tian-jiao Chen,Ping Zhu et al. (2014). The effect of high-pressure homogenized engineered yeast cells,Low temperature preservation and de glycosylation treatment On theactivity of LXYL-P1-2.Chinese Medical Biotechnology ,9(5):381-384.
Yan Li，Nan Ruan,Cui-zhu Yang et al. (2008).Effects of differrent methods of breaking cells wall on yeast RNA extraction. Food Science, 29(3): 204-206.
Zhi-guo Su et al. (1990).Extraction of intrecellularproteins from yeast by disruption in a high pressure homogenizer. Chinese journalof Biotechnology, 1990,6 (3):240-245.
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