Quality and authenticity of heterologous proteins synthesized in yeast

Eckart, Michael; Bussineau, C. M.

doi:10.1016/s0958-1669(96)80056-5

Cited by 98 publications

(59 citation statements)

References 43 publications

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“…First, a mammalian PKC␣ expressed in the yeast may not be properly post-translationally modified. For example, recombinant glycoproteins expressed in S. cerevisiae are of the high mannose type and could be hyperglycosylated (56). Second, Su et al (9) used crude yeast lysate containing the ectopically expressed PKC as the source of the enzyme although we used the immunoprecipitated PKC␣ in the in vitro kinase assays.…”

Section: Discussionmentioning

confidence: 99%

The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

Yeong

Zhu

Smith

et al. 2006

Journal of Biological Chemistry

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The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKC␣ is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ϳ60% of the catalytic activity of the mutant PKC␣, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKC␣ in immune complex kinase assays. The PKC␣ C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKC␣ immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKC␣ immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKC␣ is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKC␣ function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

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Section: Discussionmentioning

confidence: 99%

The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

Yeong

Zhu

Smith

et al. 2006

Journal of Biological Chemistry

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“…The genetic background of a Pichia host strain can influence the level of transcription, translation efficiency, the secretory pathway, protein quality, plasmid stability and plasmid copy number [34]. This is best illustrated by the use of protease-deficient strains to improve the quality and yields of various heterologous proteins [111].…”

Section: Controlling Proteolysismentioning

confidence: 99%

“…It is also one of the more complex [34]. It is thought that since many mammalian native proteins are glycosylated, it must be necessary to have the correct glycosylation patterns on recombinant proteins to ensure their biological activity.…”

Section: O-and N-linked Glycosylationmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,097

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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“…Like mammalian cells, yeast also use GPCRs and G-proteins naturally to communicate with their environment (Pausch, 1997). Also, they have the ability to perform most post-translational protein modifications, which may be essential for proper protein function (Eckart and Bussineau, 1996). Specifically, for the expression of GPCRs in yeast, advances have made use of sequence information gathered from the endogenous STE2 yeast receptor, in order to properly target the protein to the membrane (Reilander and Weiss, 1998;Sarramegna et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

O’Malley

Lazarova

Britton

et al. 2007

Journal of Structural Biology

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The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A 2 a receptor fused to a green fluorescent protein (A 2 aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A 2 aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A 2 aR and A 2 aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A 2 aR-GFP-His 10 . One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A 2 aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A 2 a-His 10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A 2 aR yet achieved from any heterologous expression system.

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Quality and authenticity of heterologous proteins synthesized in yeast

Cited by 98 publications

References 43 publications

The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

The Last 10 Amino Acid Residues beyond the Hydrophobic Motif Are Critical for the Catalytic Competence and Function of Protein Kinase Cα

Heterologous protein production using the Pichia pastoris expression system

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

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