scholarly journals Engineering Saccharomyces Cerevisiae for the Production and Secretion of Affibody Molecules.

2021 ◽  
Author(s):  
Veronica Gast ◽  
Anna Sandegren ◽  
Finn Dunås ◽  
Siri Ekblad ◽  
Rezan Güler ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carboxypeptidase Y ◽  
Affibody Molecules ◽  
E Coli ◽  
Fed Batch Fermentation ◽  
Proteinase A ◽  
Production Host ◽  
Diagnostic Applications ◽  
High Yields

Abstract BackgroundAffibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to address if improvements in terms of yield, ease of production and if purification advantages can be identified. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, showed to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind human HER3 as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. ConclusionThis study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching high yields and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

scholarly journals Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

2013 ◽  
Vol 288 (20) ◽  
pp. 13951-13959 ◽  
Author(s):  
Yan Zhang ◽  
Xiuxiang An ◽  
JoAnne Stubbe ◽  
Mingxia Huang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Small Subunit ◽  
Cluster Assembly ◽  
E Coli ◽  
Viability Assay ◽  
Docking Model

The small subunit (β2) of class Ia ribonucleotide reductase (RNR) houses a diferric tyrosyl cofactor (Fe2III-Y•) that initiates nucleotide reduction in the large subunit (α2) via a long range radical transfer (RT) pathway in the holo-(α2)m(β2)n complex. The C-terminal tails of β2 are predominantly responsible for interaction with α2, with a conserved tyrosine residue in the tail (Tyr356 in Escherichia coli NrdB) proposed to participate in cofactor assembly/maintenance and in RT. In the absence of structure of any holo-RNR, the role of the β tail in cluster assembly/maintenance and its predisposition within the holo-complex have remained unknown. In this study, we have taken advantage of the unusual heterodimeric nature of the Saccharomyces cerevisiae RNR small subunit (ββ′), of which only β contains a cofactor, to address both of these issues. We demonstrate that neither β-Tyr376 nor β′-Tyr323 (Tyr356 equivalent in NrdB) is required for cofactor assembly in vivo, in contrast to the previously proposed mechanism for E. coli cofactor maintenance and assembly in vitro. Furthermore, studies with reconstituted-ββ′ and an in vivo viability assay show that β-Tyr376 is essential for RT, whereas Tyr323 in β′ is not. Although the C-terminal tail of β′ is dispensable for cofactor formation and RT, it is essential for interactions with β and α to form the active holo-RNR. Together the results provide the first evidence of a directed orientation of the β and β′ C-terminal tails relative to α within the holoenzyme consistent with a docking model of the two subunits and argue against RT across the β β′ interface.

Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1191-1199
Author(s):  
M Bernstein ◽  
F Kepes ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Product ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Partial Restoration ◽  
Alpha Factor ◽  
Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

scholarly journals The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis

1999 ◽  
Vol 338 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Evelyne RAUX ◽  
Treasa McVEIGH ◽  
Sarah E. PETERS ◽  
Thomas LEUSTEK ◽  
Martin J. WARREN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Biosynthetic Pathways ◽  
Subtle Difference ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
His Tag ◽  
Cobalamin Biosynthesis

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.

scholarly journals Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (3) ◽  
pp. 1191-1199 ◽  
Author(s):  
M Bernstein ◽  
F Kepes ◽  
R Schekman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Product ◽  
Carboxypeptidase Y ◽  
Secretory Proteins ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Partial Restoration ◽  
Alpha Factor ◽  
Mutant Cells

When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins. Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent. Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons. This prediction was confirmed by reconstitution of the sec59 defect in vitro. The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells. Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes. Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides. These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

scholarly journals Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli

1989 ◽  
Vol 9 (11) ◽  
pp. 4767-4776
Author(s):  
G B Sancar ◽  
F W Smith
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Yeast Cells ◽  
E Coli ◽  
Pyrimidine Dimers ◽  
Nucleotide Excision ◽  
Dark Survival

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.

Optimization of Dengue-3 recombinant NS1 protein expression in E. coli and in vitro refolding for diagnostic applications

Virus Genes ◽  
2012 ◽  
Vol 46 (2) ◽  
pp. 219-230 ◽  
Author(s):  
T. N. Athmaram ◽  
Shweta Saraswat ◽  
Princi Misra ◽  
Saurabh Shrivastava ◽  
Anil K. Singh ◽  
...  
Keyword(s):  
Protein Expression ◽  
Ns1 Protein ◽  
E Coli ◽  
Diagnostic Applications ◽  
In Vitro Refolding

scholarly journals THE INABILITY OF YEAST TRANSFER RNA TO SUPPRESS AN AMBER MUTATION IN AN E. coli SYSTEM

Genetics ◽  
1973 ◽  
Vol 73 (1) ◽  
pp. 23-28
Author(s):  
John A Kiger ◽  
Carol J Brantner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transfer Rna ◽  
Amber Mutation ◽  
E Coli ◽  
Amber Suppressor ◽  
Suppressor Mutants

ABSTRACT Transfer RNA from super-suppressor mutants of Saccharomyces cerevisiae cannot suppress an amber mutation in vitro in an E. coli protein synthesizing system. It is tentatively concluded that the yeast amber suppressor does not contain a transfer RNA altered in the anticodon.

scholarly journals Prepro-carboxypeptidase Y and a truncated form of pre-invertase, but not full-length pre-invertase, can be posttranslationally translocated across microsomal vesicle membranes from Saccharomyces cerevisiae.

1988 ◽  
Vol 106 (4) ◽  
pp. 1075-1081 ◽  
Author(s):  
W Hansen ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Full Length ◽  
Microsomal Membrane ◽  
Carboxypeptidase Y ◽  
Truncated Form ◽  
In Vitro System ◽  
Vesicle Membranes

We have determined that prepro-carboxypeptidase Y and a truncated form of pre-invertase can be translocated across the yeast microsomal membrane post-translationally in a homologous in vitro system. The yeast secretory protein prepro-alpha-factor which was previously shown to be an efficient posttranslational translocation substrate is therefore not unique in this regard, but rather the yeast ER protein translocation machinery is generally capable of accepting substrates from a ribosome-free, soluble pool. However, within our detection limits, full-length pre-invertase could not be translocated posttranslationally, but was translocated co-translationally. This indicates that not every fully synthesized pre-protein can use this pathway, presumably because normal or aberrant folding characteristics can interfere with translocation competence.

scholarly journals Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent.

1987 ◽  
Vol 105 (6) ◽  
pp. 2905-2914 ◽  
Author(s):  
P Bird ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Influenza Virus ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Signal Sequence ◽  
Carboxypeptidase Y ◽  
Influenza Virus Hemagglutinin

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed.

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