Impact of lipopolysaccharides on cultivation and recombinant protein expression in human embryonal kidney (HEK‐293) cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.

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Influence of glutamine on transient and stable recombinant protein production in CHO and HEK-293 cells

BMC Proceedings ◽

10.1186/1753-6561-5-s8-p35 ◽

2011 ◽

Vol 5 (Suppl 8) ◽

pp. P35 ◽

Cited By ~ 1

Author(s):

Yashas Rajendra ◽

Divor Kiseljak ◽

Lucia Baldi ◽

David L Hacker ◽

Florian M Wurm

Keyword(s):

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Regulation of LIM-domain-binding 1 protein expression by ubiquitination of Lys134

Biochemical Journal ◽

10.1042/bj20091461 ◽

2010 ◽

Vol 429 (1) ◽

pp. 127-136 ◽

Cited By ~ 4

Author(s):

Paul W. Howard ◽

Shall F. Jue ◽

David G. Ransom ◽

Richard A. Maurer

Keyword(s):

Protein Expression ◽

Ring Finger ◽

Lim Domain ◽

Hek 293 ◽

Hek 293 Cells ◽

Lim Domains ◽

293 Cells ◽

Transcriptional Regulatory ◽

Enhanced Expression ◽

Proteasome Pathway

LDB1 (LIM-domain-binding 1) is a cofactor that participates in formation of transcriptional regulatory complexes involving transcription factors containing LIM domains as well as other factors. The amount of LDB1 protein in cells has previously been shown to be modulated by RNF12 (RING finger protein 12). RNF12 is an E3 ubiquitin ligase that can target LDB1 for poly-ubiquitination and degradation via the proteasome. We find that in HEK (human embryonic kidney)-293 cells expression of RNF12 leads to mono-ubiquitination of LDB1 and increased levels of LDB1 protein. Mutagenesis studies identified Lys134 of LDB1 as the residue that is mono-ubiquitinated by RNF12. Mutation of Lys134 of LDB1 to arginine blocks the formation of mono-ubiquitinated LDB1 and surprisingly also increases LDB1 protein expression in HEK-293 cells. This leads to a model in which Lys134 of LDB1 can be either mono-ubiquitinated, leading to stabilization, or poly-ubiquitinated, leading to degradation by the proteasome pathway. We also find that ubiquitin–LDB1 fusion proteins are stabilized in HEK-293 cells, offering further evidence that mono-ubiquitination stabilizes LDB1 in these cells. Expression in Xenopus laevis embryos of an LDB1 protein in which Lys134 is replaced with arginine leads to enhanced expression of the mutant protein as compared with the wild-type protein. These findings provide evidence that modification of Lys134 can play a major role in regulating LDB1 expression.

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