Repressing expression of difficult-to-express recombinant proteins during the selection process increases productivity of CHO stable pools

More than half of licensed therapeutic recombinant proteins (r-proteins) are manufactured using constitutively-expressing, stably-transfected Chinese hamster ovary (CHO) clones. While constitutive CHO expression systems have proven their efficacy for the manufacturing of monoclonal antibodies, many next-generation therapeutics such as cytokines and bispecific antibodies as well as biological targets such as ectodomains of transmembrane receptors remain intrinsically challenging to produce. Herein, we exploited a cumate-inducible CHO platform allowing reduced expression of various classes of r-proteins during selection of stable pools. Following stable pool generation, fed-batch productions showed that pools generated without cumate (OFF-pools) were significantly more productive than pools selected in the presence of cumate (ON-pools) for 8 out of the 10 r-proteins tested, including cytokines, G-protein coupled receptors (GPCRs), the HVEM membrane receptor ectodomain, the multifunctional protein High Mobility Group protein B1 (HMGB1), as well as monoclonal and bispecific T-cell engager antibodies. We showed that OFF-pools contain a significantly larger proportion of cells producing high levels of r-proteins and that these cells tend to proliferate faster when expression is turned off, suggesting that r-protein overexpression imposes a metabolic burden on the cells. Cell viability was lower and pool recovery was delayed during selection of ON-pools (mimicking constitutive expression), suggesting that high producers were likely lost or overgrown by faster-growing, low-producing cells. We also observed a correlation between the expression levels of the GPCRs with Binding immunoglobulin Protein, an endoplasmic reticulum (ER) stress marker. Taken together, these data suggest that using an inducible system to minimize r-protein expression during stable CHO pool selection reduces cellular stresses, including ER stress and metabolic burden, leading to pools with greater frequency of high-expressing cells, resulting in improved volumetric productivity.     

Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.     

Galactosylation variations in marketed therapeutic antibodies

There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.     

Cross-genomic analysis of the translational systems of various organisms

We have characterized the genes encoding ribosomal proteins (r-proteins) as well as other translation-related factors of 15 eubacteria and four archaebacteria, and the genes for the mitochondrial r-proteins of Saccharomyces cerevisiae by using the complete genomic nucleotide sequence data of these organisms. In eubacteria, including two species of Mycoplasma, the operon structure of the r-protein genes is well conserved, while their relative orientation and chromosomal location are quite divergent. The operon structure of the r-protein genes in archaebacteria, on the other hand, is quite different from eubacteria and also among themselves. In addition, many archaebacterial r-proteins show similarity to rat cytoplasmic r-proteins. Nonetheless, characteristic features of several genes encoding proteins of functional importance are well conserved throughout the bacterial species including archaebacteria, as well as in S. cerevisiae. We searched for the genes encoding mitochondrial r-proteins in yeast by combining informatics and genetic experiments. Furthermore, we characterized some of the r-proteins genes by exchanging portions between Escherichia coli and S. cerevisiae and performed functional analysis of some of the genes from different evolutionary points of view. Our work may be extended towards phylogenetic analysis of organisms producing secondary metabolites of various sorts. Journal of Industrial Microbiology & Biotechnology (2001) 27, 163–169. Received 21 September 1999/ Accepted in revised form 22 September 2000     

The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.     

Evolution of proteins in mammalian cytoplasmic and mitochondrial ribosomes

Summary The proteins of cytoplasmic and mitochondrial ribosomes from the cow and the rat were analyzed by co-electrophoresis in two dimensional polyacrylamide gels to determine their relative evolutionary rates. In a pairwise comparison of individual ribosomal proteins (r-proteins) from the cow and the rat, over 85% of the cytoplasmic r-proteins have conserved electrophoretic properties in this system, while only 15% of the proteins of mitochondrial ribosomes from these animals fell into this category. These values predict that mammalian mitochondrial r-proteins are evolving about 13 times more rapidly than cytoplasmic r-proteins. Based on actual evolutionary rates for representative cytoplasmic r-proteins, this mitochondrial r-protein evolutionary rate corresponds to an amino acid substitution rate of 40×10^–10 per site per year, placing mitochondrial r-proteins in the category of rapidly evolving proteins. The mitochondrial r-proteins are apparently evolving at a rate comparable to that of the mitochondrial rRNA, suggesting that functional constraints act more or less equally on both kinds of molecules in the ribosome. It is significant that mammalian mitochondrial r-proteins are evolving more rapidly than cytoplasmic r-proteins in the same cell, since both sets of r-proteins are encoded by nuclear genes. Such a difference in evolutionary rates implies that the functional constraints operating on ribosomes are somewhat relaxed for mitochondrial ribosomes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.     

The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome

Eukaryotic ribosomes are made of two components, four ribosomal RNAs, and approximately 80 ribosomal proteins (r-proteins). The exact number of r-proteins and r-protein genes in higher plants is not known. The strong conservation in eukaryotic r-protein primary sequence allowed us to use the well-characterized rat (Rattus norvegicus) r-protein set to identify orthologues on the five haploid chromosomes of Arabidopsis. By use of the numerous expressed sequence tag (EST) accessions and the complete genomic sequence of this species, we identified 249 genes (including some pseudogenes) corresponding to 80 (32 small subunit and 48 large subunit) cytoplasmic r-protein types. None of the r-protein genes are single copy and most are encoded by three or four expressed genes, indicative of the internal duplication of the Arabidopsis genome. The r-proteins are distributed throughout the genome. Inspection of genes in the vicinity of r-protein gene family members confirms extensive duplications of large chromosome fragments and sheds light on the evolutionary history of the Arabidopsis genome. Examination of large duplicated regions indicated that a significant fraction of the r-protein genes have been either lost from one of the duplicated fragments or inserted after the initial duplication event. Only 52 r-protein genes lack a matching EST accession, and 19 of these contain incomplete open reading frames, confirming that most genes are expressed. Assessment of cognate EST numbers suggests that r-protein gene family members are differentially expressed.     

Transient gene expression in suspension HEK-293 cells: application to large-scale protein production

Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.     

Phylogenomics of prokaryotic ribosomal proteins

Archaeal and bacterial ribosomes contain more than 50 proteins, including 34 that are universally conserved in the three domains of cellular life (bacteria, archaea, and eukaryotes). Despite the high sequence conservation, annotation of ribosomal (r-) protein genes is often difficult because of their short lengths and biased sequence composition. We developed an automated computational pipeline for identification of r-protein genes and applied it to 995 completely sequenced bacterial and 87 archaeal genomes available in the RefSeq database. The pipeline employs curated seed alignments of r-proteins to run position-specific scoring matrix (PSSM)-based BLAST searches against six-frame genome translations, mitigating possible gene annotation errors. As a result of this analysis, we performed a census of prokaryotic r-protein complements, enumerated missing and paralogous r-proteins, and analyzed the distributions of ribosomal protein genes among chromosomal partitions. Phyletic patterns of bacterial and archaeal r-protein genes were mapped to phylogenetic trees reconstructed from concatenated alignments of r-proteins to reveal the history of likely multiple independent gains and losses. These alignments, available for download, can be used as search profiles to improve genome annotation of r-proteins and for further comparative genomics studies.     

Stepwise Splitting of Ribosomal Proteins from Yeast Ribosomes by LiCl

Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5–1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.     

Changes in regulation of ribosomal protein synthesis during vegetative growth and sporulation of Saccharomyces cerevisiae.

When diploid Saccharomyces cerevisiae cells logarithmically growing in acetate medium were placed in sporulation medium, the relative rates of synthesis of 40 or more individual ribosomal proteins (r-proteins) were coordinately depressed to approximately 20% of those of growing cells. These new depressed rates remained constant for at least 10 h into sporulation. If yeast nitrogen base was added 4 yh after the beginning of sporulation to shift the cells back to vegetative growth, the original relative rates of r-protein synthesis were rapidly reestablished. this upshift in the rates occurred even in diploids homozygous for the regulatory mutation rna2 at the restrictive temperature for this mutation (34 degrees C). However, once these mutant cells began to bud and grow at 34 degrees C, the phenotype of rna2 was expressed and the syntheses of r-proteins were again coordinately depressed. At least one protein whose rate of synthesis was not depressed by rna2 in vegetative cells did have a decreased rate of synthesis during sporulation. Another r-protein whose synthesis was depressed by rna2 maintained a high rate of synthesis at the beginning of sporulation. These data suggest that the mechanism responsible for coordinate control of r-protein synthesis during sporulation does not require the gene product of RNA2 and thus defines a separate mechanism by which r-proteins are coordinately controlled in S. cerevisiae.     

Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed.     

Herpes simplex virus type 1 strain HSV1716 grown in baby hamster kidney cells has altered tropism for nonpermissive Chinese hamster ovary cells compared to HSV1716 grown in vero cells

Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.     

Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.     

Histone deacetylases control lysine acetylation of ribosomal proteins in rice

Lysine acetylation (Kac) is well known to occur in histones for chromatin function and epigenetic regulation. In addition to histones, Kac is also detected in a large number of proteins with diverse biological functions. However, Kac function and regulatory mechanism for most proteins are unclear. In this work, we studied mutation effects of rice genes encoding cytoplasm-localized histone deacetylases (HDAC) on protein acetylome and found that the HDAC protein HDA714 was a major deacetylase of the rice non-histone proteins including many ribosomal proteins (r-proteins) and translation factors that were extensively acetylated. HDA714 loss-of-function mutations increased Kac levels but reduced abundance of r-proteins. In vitro and in vivo experiments showed that HDA714 interacted with r-proteins and reduced their Kac. Substitutions of lysine by arginine (depleting Kac) in several r-proteins enhance, while mutations of lysine to glutamine (mimicking Kac) decrease their stability in transient expression system. Ribo-seq analysis revealed that the hda714 mutations resulted in increased ribosome stalling frequency. Collectively, the results uncover Kac as a functional posttranslational modification of r-proteins which is controlled by histone deacetylases, extending the role of Kac in gene expression to protein translational regulation.