Evidence map›Paper›PMID 33115654›Full record

ArticleProtein expression and purification2021

Expression and purification of active human kinases using Pichia pastoris as a general-purpose host.

May H Abdel Aziz, Yao Fan, Lijun Liu, Mark M Moasser, Haian Fu, Natalia Jura, Michelle R Arkin

Open access · greenAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Protein expression and purification, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.

0numbers the graph read from it
0cells of the map it votes in
1citing papers in PubMed
0.1field-weighted citation impact, top 55% of its field
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

1 citing paper in PubMed, 1 citations in OpenAlex.

  1. Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

7 authors at 2 institutions in 1 country.

May H Abdel AzizSmall Molecule Discovery Center, Department of Pharmaceutical Chemistry and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA. Electronic address: mabdelaziz@uttyler.edu.
Yao FanSmall Molecule Discovery Center, Department of Pharmaceutical Chemistry and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
Lijun LiuCardiovascular Research Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.
Mark M MoasserDepartment of Medicine and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
Haian FuDepartment of Pharmacology and Chemical Biology and Emory Chemical Biology Discovery Center, Emory University School of Medicine and Winship Cancer Institute, Atlanta, GA, USA.
Natalia JuraCardiovascular Research Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.
Michelle R ArkinSmall Molecule Discovery Center, Department of Pharmaceutical Chemistry and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA. Electronic address: michelle.arkin@ucsf.edu.
University of California, San Francisco · USEmory University · US

Funding

Understanding resistance to HER family tyrosine kinase inhibitorsR01CA122216 · NCI · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · PI MOASSER, MARK M · 2007 to 2017
$3.0M
NCI NIH HHS R01 CA122216
6 · The paper itself

Abstract

backgroundThe 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.

methodsSix 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. The standard expression procedure for P. pastoris was adopted, followed by purification using affinity chromatography. Purity and activity of the proteins were confirmed and compared to published values.

resultsSome 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.

Indexed as

Protein KinasesRecombinant Fusion ProteinsSaccharomycetalesAnimalsChromatography, AffinityHumansProtein DomainsSf9 CellsSolubilityProtein KinasesRecombinant Fusion ProteinsActivityExpressionKinaseP. pastorisYeast

Identifiers

PMID33115654
PMCPMC11655031
OpenAlexW3087612654

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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.