Optimization of conditions for the productionof Hsp70 phaperones in Saccharomyces cerevisiae pells

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Abstract

BACKGROUND: Molecular chaperones regulate the proper folding of proteins in the cell. Members of the Hsp70 family, including the Ssa1 protein, are molecular chaperones that prevent protein aggregation, promote their proper folding and degradation, and are the most common among the various chaperones, highly conserved, and present in a variety of organisms.

AIM: The aim of the work was to optimize methods for the production, extraction and purification of Ssa1 protein from cells of Saccharomyces cerevisiae.

MATERIALS AND METHODS: The SSA1-4 gene sequences were cloned into a vector under the control of the TEF1 promoter and fused with a sequence encoding His6-tag. Yeast strains with different genetic backgrounds were transformed with the obtained constructs, and the production of Ssa1-4 proteins was assessed under different cultivation conditions. Affinity and ion-exchange chromatography were used to purify the Ssa1 protein. Fluorescence microscopy was used to confirm the localization of recombinant Ssa proteins fused with TagRFP-T in the cytosol.

RESULTS AND CONCLUSIONS: Methods for the production, extraction and purification of Ssa1 protein from yeast cells have been optimized. The same approach can be further used to purify other Hsp70 proteins and adapted to obtain various proteins from eukaryotic cells.

About the authors

Andrew G. Matveenko

Saint Petersburg State University

Email: a.matveenko@spbu.ru
ORCID iD: 0000-0002-9458-0194
SPIN-code: 9877-5352

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Andrew A. Tsvetkov

Saint Petersburg State University

Email: st096303@student.spbu.ru
ORCID iD: 0009-0007-3673-7310
Russian Federation, Saint Petersburg

Tatyana M. Rogoza

Saint Petersburg State University; St. Petersburg Branch, Vavilov Institute of General Genetics of the Russian Academy of Sciences

Email: t.rogoza@spbu.ru
ORCID iD: 0000-0003-2981-0421
SPIN-code: 7582-1519

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg; Saint Petersburg

Yury A. Barbitoff

Saint Petersburg State University

Email: barbitoff@bk.ru
ORCID iD: 0000-0002-3222-440X
SPIN-code: 1053-6164

Cand. Sci. (Biology)

Russian Federation, Saint Petersburg

Galina A. Zhouravleva

Saint Petersburg State University

Author for correspondence.
Email: g.zhuravleva@spbu.ru
ORCID iD: 0000-0002-3013-4662
SPIN-code: 3132-6884

Dr. Sci. (Biology), Professor

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Comparison of Ssa1 production levels in different yeast strains by immunoblotting. pTEF-SSA1 (a) or pTEF-SSA1-21 (b) plasmids were used for transformation. Total protein was visualized using Coomassie R250 membrane staining. log, yeast culture in logarithmic (OD₆₀₀ = 0.7–1.0), stat,in stationary (OD₆₀₀ = 2) growth phase. Anti-His6 and anti-Hsp70 antibodies were used. Strains selected for further analysis are marked with asterisks. The order of the strains in panel A is as follows (from left to right): OT56, 74-D694, P2.1.1-yAO121, yAO121, 2-yAO121, prb1Δ0-P-74-D694,prb1Δ0-2-74-D694, yAO066, prb1Δ-BY4741, BY4742, yAO066 (stat), prb1Δ-BY4741(stat), BY4742(stat). The order of the strains in panel B is as follows (from left to right): OT56, 74-D694, P2.1.1-yAO121, yAO121, 2-yAO121, prb1Δ0-P-74-D694, prb1Δ0-2-74-D694, prb1Δ-BY4741, BY4742, yAO066 (stat), prb1Δ-BY4741(stat), BY4742(stat).

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3. Fig. 2. Comparison of Ssa1 production levels in the selected strains under different conditions: a, yAO121 and prb1Δ-BY4741 transformants fromfig. 1 were first grown in 30°C to mid-log phase (OD₆₀₀ = 0.4–0.6), and then incubated at the indicated temperatures for 4 hours: b, Strain prb1Δ-BY4741 was transformed with pTEF-SSA1, pTEF-SSA1-21, or pRS316 (e.v., empty vector) plasmids; the selected transformants were grown at SC-Ura (SC)or Min-Ura (Min) media at 30°C to the late-log phase. Two independent transformants are shown in each case. Shown are results of western blot analysis. Total protein was visualized using Coomassie R250 membrane staining.

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4. Fig. 3. Selection of a medium for the production of Ssa2, Ssa3 and Ssa 4. Strain prb1Δ-BY4741 was transformed with pTEF-His6-SSA2,pTEF-His6-SSA3 and pTEF-His6-SSA4; selected transformants were grown in SC-Ura (SC) or Min-Ura (Min) media at 30°C until OD₆₀₀ = 0.8–1.1,and then subjected to protein extraction and western blotting.

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5. Fig. 4. The analysis of intracellular distribution of Ssa proteins. OT56 ([PSI⁺]) and 74-D694 ([psi⁻]) strains were transformed with a series of pTEF-His₆-yTagRFP-T-SSA plasmids. The resulting transformants were analyzed using fluorescence microscopy. BF, bright field.

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6. Fig. 5. Purification of the Ssa1 protein from yeast cell lysates: a, SDS-PAGE of affinity chromatography fractions of yeast lysates producing His₆-Ssa1 under non-denaturing conditions. Shown is Coomassie R250 staining of gels loaded with elution fractions. Fractions that were taken for subsequent concentration and purification by ion exchange chromatography are marked with a line, fractions marked with one and two arrows were used for the immunoblotting; b, Western blot of representative fractions (marked with one and two arrows at the panel A) after affinity purification; c, SDS-PAGE analysis of ion exchange chromatography fractions, stained with Coomassie R250.

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