Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones

Abstract

Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred as monomeric in this work) in a straightforward and facile manner will obviate protein –production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.

Microarray analysis of the transcriptome of theEscherichia coli(E. coli) regulated by cinnamaldehyde (CMA)

“…They are thought to assist in the folding of their target proteins by providing a sequestered environment which is conducive to correct folding. In this environment, the extended proteins can fold and shield from nonproductive interactions with other proteins (Grigoroudis, McInnes, Premnathc, & Kontopidis, 2015). In this study, two dna genes encoding chaperonins, dnaK and groEL, were induced by CMA.…”

Expression of codon-optimized TgMIC16 in three Escherichia coli strains

“…However, in this study, all three expression strains were unable to express the recombinant protein as a soluble product at either 37 or 15°C. Formation of inclusion bodies represent a complex and dynamic process affected by various factors, with no universal approach for the efficient production of soluble forms of aggregation-prone recombinant proteins (Baneyx and Mujacic 2004;Sørensen and Mortensen 2005;Grigoroudis et al 2015). Several strategies, including introduction of the expression of molecular chaperones in host bacteria, modulation of target-protein expression levels, optimization of culture medium, and addition of exogenous chaperones, might potentially prevent rTgMIC16 recombinant aggregation and will be tested in future studies.…”

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

“…Co-expression with several types of chaperones involved in protein folding in vivo is one of the approaches used to improve the solubility of the recombinant protein (Grigoroudis et al, 2015;Paraskevopoulou & Falcone, 2018;Peng et al, 2016;Wang et al, 2018). Nevertheless, to date only one study has been conducted where they co-expressed chaperones together with L-ASNase (Biglari Goliloo et al, 2021).Biglari Goliloo et al ( 2021) assessed the yield of the co-expression of the GroELS/TF system in the production of L-ASNase (Q59LAsp).…”