scholarly journals The effect of cAMP and cGMP on the activity and substrate specificity of protein kinase A from methylotrophic yeast Pichia pastoris.

2003 ◽  
Vol 50 (4) ◽  
pp. 1111-1118 ◽  
Author(s):  
Magdalena Frajnt ◽  
Małgorzata Cytryńska ◽  
Teresa Jakubowicz
Keyword(s):  
Protein Kinase ◽  
Pichia Pastoris ◽  
Pyruvate Kinase ◽  
Methylotrophic Yeast ◽  
Yeast Cells ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Ribosomal Protein S6 ◽  
Pichia Pastoris Yeast ◽  
Dependent Protein ◽  
Camp And Cgmp

Cyclic AMP dependent protein kinase (PKA) from Pichia pastoris yeast cells was found to be activated by either cAMP or cGMP. Analogs of cAMP such as 8-chloro-cAMP and 8-bromo-cAMP were as potent as cAMP in PKA activation while N6,2'-O-dibutyryl-cAMP did not stimulate the enzyme activity. It was shown that protamine sulfate was almost equally phosphorylated in the presence of 1-2 x 10(-6)M cAMP or cGMP while other substrates such as Kemptide, ribosomal protein S6, were phosphorylated to a lower extent in the presence of cGMP. It was demonstrated that pyruvate kinase is a substrate of PKA which co-purified with the P.pastoris enzyme. Moreover, pyruvate kinase was phosphorylated by PKA in the presence of cAMP and cGMP to comparable levels.

scholarly journals Expression and Purification of Active Human Kinases Using Pichia pastoris as A General-Purpose Host

2020 ◽  
Author(s):  
May H. Abdel Aziz ◽  
Yao Fan ◽  
Lijun Liu ◽  
Mark Moasser ◽  
Haian Fu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Pichia Pastoris ◽  
Protein Interactions ◽  
Active Form ◽  
Methylotrophic Yeast ◽  
Aurora Kinase ◽  
General Purpose ◽  
Mitogen Activated Protein Kinase ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Mitogen Activated Protein

Abstract Background: The heterologous expression of human kinases in good purity and in a monomeric, soluble and active form can be challenging. Most of the reported successful attempts are carried out in insect cells as a host. The use of E. coli for expression is limited to a few kinases and usually is facilitated by large solubility tags that can limit biophysical studies and affect protein–protein interactions. In this report, we evaluate the methylotrophic yeast Pichia pastoris (P. pastoris) as a general-purpose host for expression of human kinases. Methods: Six diverse kinases were chosen due to their therapeutic importance in human cancers. Tested proteins include serine/threonine kinases cyclin-dependent kinases 4 and 6 (CDK4 and 6) and aurora kinase A (AurKA), receptor tyrosine kinase erbB-2 (HER2), and dual specificity kinase mitogen-activated protein kinase kinase 3 (MKK3b). Noting that positively charged kinases expressed with higher yield, we sought to improve expression of two challenging targets, CDK6 and HER2, by fusing the highly basic, N-terminal domain of the secreted tyrosine-protein kinase VLK. A standard expression procedure was developed for P. pastoris, followed by purification using affinity chromatography. Purity and activity of the proteins were confirmed and compared to published values. Results: Some kinases were purified with good yield and purity and with comparable activity to commercially available versions. Addition of the VLK domain improved expression and decreased aggregation of CDK6 and HER2. Conclusions: P. pastoris is a promising host for expression of soluble and active human kinases.

scholarly journals Expression and Purification of Active Human Kinases Using Pichia pastoris as A General-Purpose Host

2019 ◽  
Author(s):  
May H. Abdel Aziz ◽  
Yao Fan ◽  
Lijun Liu ◽  
Mark Moasser ◽  
Haian Fu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Pichia Pastoris ◽  
Protein Interactions ◽  
Active Form ◽  
Methylotrophic Yeast ◽  
Aurora Kinase ◽  
General Purpose ◽  
Mitogen Activated Protein Kinase ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Mitogen Activated Protein

Abstract The heterologous expression of human kinases in good purity and monomer, soluble and active form can become a challenging feat. Most of the reported successful attempts are carried out in insect cells as a host. The use of E. coli for expression is limited to a few kinases and usually is facilitated by large solubility tags that can limit biophysical studies and affect protein–protein interactions. In this report, we evaluate the methylotrophic yeast Pichia pastoris as a general-purpose host for expression of human kinases. Methods: Six diverse kinases were chosen due to their therapeutic importance in human cancers. Tested proteins include serine/threonine kinases cyclin-dependent kinases 4 and 6 (CDK4 and 6) and aurora kinase A (AurKA), receptor tyrosine kinase erbB-2 (HER2), and dual specificity kinase mitogen-activated protein kinase kinase 3 (MKK3b). Two challenging targets, CDK6 and HER2, were fused to the highly basic, N-terminal domain of the secreted tyrosine-protein kinase VLK. Standard expression procedure in Pichia pastoris used. Purification was done using affinity chromatography, purity and identity of the proteins were confirmed and compared. Results: Some of the tested kinases expressed with good yield and purity and comparable activity to commercially available versins. Addition of the VLK domain improved expression and decreased aggregation of two of the challenging CDK6 and HER2. This can be attributed to a correlation we observed between the protein pI and its expression level in P. pastoris, where basic proteins are more likely to be expressed and purified. Conclusions: This study introduces P. pastoris as a promising host for expression of soluble and active human kinases.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

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