scholarly journals Protein Expression Both in Mammalian Cell Lines and in Yeast Pichia pastoris Using a Single Expression Plasmid

BioTechniques ◽  
1998 ◽  
Vol 24 (2) ◽  
pp. 266-271 ◽  
Author(s):  
Zhong Liu ◽  
Linda M. Cashion ◽  
Haifeng Pu
Keyword(s):  
Pichia Pastoris ◽  
Protein Expression ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Expression Plasmid ◽  
Yeast Pichia Pastoris ◽  
Mammalian Cell Lines

Analysis of Protein Expression and Virus-like Particle Formation in Mammalian Cell Lines Stably Expressing HIV-1 gag and env Gene Products with or without Active HIV Proteinase

Virology ◽  
1993 ◽  
Vol 192 (2) ◽  
pp. 605-617 ◽  
Author(s):  
Hans Georg Kräusslich ◽  
Christina Ochsenbauer ◽  
Anke-Mareil Traenckner ◽  
Klaus Mergener ◽  
Michael Fäcke ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Particle Formation ◽  
Gene Products ◽  
Mammalian Cell Lines ◽  
Virus Like Particle ◽  
Hiv 1

Vector and Cell Line Engineering Technologies Toward Recombinant Protein Expression in Mammalian Cell Lines

2018 ◽  
Vol 185 (4) ◽  
pp. 986-1003 ◽  
Author(s):  
Seyedeh Hoda Jazayeri ◽  
Amir Amiri-Yekta ◽  
Salahadin Bahrami ◽  
Hamid Gourabi ◽  
Mohammad Hossein Sanati ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Protein Expression ◽  
Mammalian Cell Lines ◽  
Line Engineering

scholarly journals Placing the RPL32 Promoter Upstream of a Second Promoter Results in a Strongly Increased Number of Stably Transfected Mammalian Cell Lines That Display High Protein Expression Levels

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
F. Hoeksema ◽  
K. Hamer ◽  
M. Siep ◽  
J. A. Verhees ◽  
A. P. Otte
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Small Region ◽  
High Protein ◽  
Expression Levels ◽  
Mammalian Cell Lines ◽  
Selection Systems ◽  
In Cis ◽  
Gene Expression Levels

The use of high stringency selection systems commonly results in a strongly diminished number of stably transfected mammalian cell lines. Here we placed twelve different promoters upstream of an adjacent primary promoter and tested whether this might result in an increased number of colonies; this is in the context of a stringent selection system. We found that only the promoter of the human ribosomal protein, RPL32, induced a high number of colonies in CHO-DG44 cells. This phenomenon was observed when the RPL32 promoter was combined with the CMV, SV40, EF1-α, and the β-actin promoters. In addition, these colonies displayed high protein expression levels. The RPL32 promoter had to be functionally intact, since the deletion of a small region upstream of the transcription start site demolished its positive action. We conclude that adding the RPL32 promoter to an expression cassette in cis may be a powerful tool to augment gene expression levels.

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

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