Engineering genes for predictable protein expression

“…RNAi has been used to target apoptosis (e.g., caspase-3 and 7 [199,200], Bak and Bax [201], requiem [184]); glycosylation (e.g., 1,6-fucosyltransferase [202,203], sialidase [204]); and enzymes such as dihydrofolate reductase (DHFR) [205,206]. Targeting gene expression can be performed using homolog recombination, a variety of nucleases, such as ZFNs, meganucleases, and TALENs [207][208][209][210]. For example, ZFNs have been used to silence expression of Bak and Bax proteins to produce apoptosis-resistant CHO cells [211].…”

Section: Applied Genomics and Cell Line Engineering In Mammalian Exprmentioning

confidence: 99%

Emerging Alternative Production Systems

Sommer

Laux

Frenzel

et al. 2014

Handbook of Therapeutic Antibodies

View full text Add to dashboard Cite

The market of recombinant protein pharmaceuticals is growing rapidly. Among those, recombinant antibodies are the fastest growing group and a large number of new antibody products are in clinical and preclinical development. Since most conventional cost-effective microbial expression systems are not yet applicable for the expression of fully functional immunoglobulin G (IgG) antibodies, almost all of the currently marketed therapeutic recombinant antibody products are produced from animal cell culture processes that are associated with relative high operating expenses. Thus, to make broad use of recombinant antibody therapeutics more affordable, alternative production systems with improved efficiency and reduced operating expenses are highly desirable.IgG currently is the dominant antibody format for therapeutics. As a heterotetrameric molecule with numerous disulfide bonds and several glycosylation sites IgG makes itself a challenging candidate for heterologous expression, especially in prokaryotes. Glycosylation of the fragment crystallizable (Fc) region has been shown to be essential for mediating effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [1]. Furthermore, the glycosylation pattern strongly influences the efficiency of the induction of effector functions by antibodies [2, 3]. For applications that depend on the immunological effector functions of the antibody therefore eukaryotic production systems with suitable glycosylation capabilities are necessary.Antibody fragments such as single chain fragment variable (scFv) and fragment antigen binding (Fab) are less complex and the antigen-binding activity is independent of glycosylation, which makes them qualified for prokaryotic expression systems. Certainly, these fragments are incapable of mediating immunological effector functions but for numerous applications this is not necessary or even unwanted.Besides the two traditional expression systems, Escherichia coli for antibody fragments and Chinese hamster ovarian (CHO) or myeloma cells for IgG antibodies, numerous alternative expression systems are currently under development.

show abstract

“…mRNA secondary structures at the 5′ terminus were shown to impair translation by hindering the ribosomal binding and translational initiation (Kudla et al 2009). Moreover, the GC content and the occurrence of codons rarely used by the production host influenced translation efficiency (Gustafsson et al 2012). It was reported that especially clusters of rare codons have a detrimental effect on translation in Escherichia coli (Kane 1995) causing translational frameshifts (Spanjaard and van Duin 1988), premature translational termination (Rosenberg et al 1993), and amino acid misincorporation (Calderone et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium

Finger

Gamer

Klunkelfuß

et al. 2015

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium. For analyzing the influence of rare codons on the translational efficiency and protein production in B. megaterium, a test system using the gene for the green fluorescent protein (GFP) as reporter was established. For this purpose, four consecutive identical codons were introduced into the 5' end of gfp and the resulting variations in GFP formation were quantified. Introduction of the rare codons GCC, CGG, and ACC for alanine, arginine, and threonine reduced GFP production 2.1-, 3.3-, and 1.7-fold in comparison to the favored codons GCU, CGU, and ACA, respectively. Coexpression of the corresponding rare codon tRNA (rctRNA) genes improved GFP production 4.2-, 2.7-, and 1.7-fold, respectively. The system was applied to the production of a formate dehydrogenase (FDH) from Mycobacterium vaccae and an extracellular hydrolase (TFH) from Thermobifida fusca. Coexpression of one to three different rctRNA genes resulted in an up to 18-fold increased protein production. Interestingly, rctRNA gene coexpression also elevated the production of M. vaccae FDH and T. fusca TFH from codon optimized genes, indicating a general positive effect by rctRNA gene overexpression on the protein production in B. megaterium. Thus, the basis for a B. megaterium enhanced production strain coexpressing rctRNA genes was laid.

show abstract

Engineering genes for predictable protein expression

Cited by 154 publications

References 81 publications

Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP)

Prediction of recombinant protein overexpression in Escherichia coli using a machine learning based model (RPOLP)

Emerging Alternative Production Systems

Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium

Contact Info

Product

Resources

About