Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors.

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.

Recombinant glycoprotein product quality in proliferation-controlled BHK-21 cells.

We analyzed product quality to determine the applicability of proliferation-controlled mammalian cells for recombinant pharmaceutical protein production. Baby hamster kidney (BHK)-21 cells were engineered to express a dicistronic, stabilized, self-selecting growth control system consisting of a beta-estradiol-activatable transcription factor IRF-1 fusion protein. IRF-1 activity led to a reduced growth rate, whereas productivity, protein integrity, and glycosylation pattern of the industrially relevant secreted pharmaceutical glycoprotein erythropoietin remained consistent, showing that this technique has the potential for improving the consistency of high-quality pharmaceutical products and thus warrants further development.

Genetic optimization of recombinant glycoprotein production by mammalian cells.

Genetically modified mammalian cells are the preferred system for the production of recombinant therapeutic glycoproteins. Other applications include engineering of cell lines for drug screening and cell-based therapies, and the construction of recombinant viruses for gene therapy. This article highlights contemporary core genetic technologies and emerging strategies for genetically engineering mammalian cells for optimal recombinant-protein expression.

A ribosomal protein is required for translational regulation of GCN4 mRNA. Evidence for involvement of the ribosome in eIF2 recycling.

In amino acid-starved yeast cells, inhibition of the guanine nucleotide exchange factor eIF2B by phosphorylated translation initiation factor 2 results in increased translation of GCN4 mRNA. We isolated a suppressor of a mutant eIF2B. The suppressor prevents efficient GCN4 mRNA translation due to inactivation of the small ribosomal subunit protein Rps31 and results in low amounts of mutant 40 S ribosomal subunits. Deletion of one of two genes encoding ribosomal protein Rps17 also reduces the amounts of 40 S subunits but does not suppress eIF2B mutations or prevent efficient GCN4 translation. Our findings show that Rps31-deficient ribosomes are altered in a way that decreases the eIF2B requirement and that the small ribosomal subunit mediates the effects of low eIF2B activity on cell viability and translational regulation in response to eIF2 phosphorylation.

Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms.

We cloned the GCD5 gene of S. cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS). The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA(Lys). Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity. We propose an autoregulatory model in which uncharged tRNA(Lys) stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

Evidence for regulation of reinitiation in translational control of GCN4 mRNA.

Translational control of the GCN4 gene of Saccharomyces cerevisiae is mediated by four upstream open reading frames (URFs) present in the leader of GCN4 mRNA. URFs 3 and 4 efficiently repress GCN4 expression in normal growth conditions; URFs 1 and 2 are required to overcome this repression in amino acid-starved cells. lacZ fusions to URFs 3 and 4 were used to determine the translational event that is regulated at these sequences by URFs 1 and 2. URF3-lacZ, URF4-lacZ, and GCN4-lacZ fusions are affected similarly by URFs 1 and 2 when no other URFs are present in the leader: expression from all three fusions is reduced by an amount slightly greater in repressing than in derepressing conditions. These results are inconsistent with models that postulate a differential effect of URFs 1 and 2 on initiation or elongation rates at URFs 3 and 4 versus the GCN4 coding sequences. We propose that the efficiency of reinitiation at the GCN4 AUG codon after translation of URFs 3 and 4 is the translational event that is stimulated in derepressing conditions by URFs 1 and 2.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

A segment of GCN4 mRNA containing the upstream AUG codons confers translational control upon a heterologous yeast transcript.

GCN4 encodes a transcriptional activator in Saccharomyces cerevisiae that is regulated at the translational level. We show that an approximately 240-nucleotide segment from the GCN4 mRNA leader containing four AUG codons is sufficient to confer translational control typical of GCN4 upon a GAL1-lacZ fusion transcript. Regulation of the hybrid transcript is dependent upon multiple positive (GCN) and negative (GCD) trans-acting factors shown to regulate GCN4 expression post-transcriptionally. This result limits the target sequences for these factors to a small internal segment of the GCN4 mRNA leader. The elimination of AUG codons within this segment substantially reduces the usual derepressing effect of mutations in five GCD genes upon GCN4-lacZ expression. This supports the idea that the products of these negative regulatory genes act by modulating the effects of the upstream AUG codons on translation of GCN4 mRNA.

Multiple upstream AUG codons mediate translational control of GCN4.

GCN4 encodes a transcriptional activator of amino acid biosynthetic genes in yeast that is regulated at the translational level. The 5' leader of GCN4 mRNA contains four small open-reading-frames. By constructing point mutations in the initiation codons of these sequences, we show that they are essential for translational repression of GCN4. Each upstream AUG codon can repress translation; however, the two 3' proximal AUG codons are much more inhibitory than the 5' proximal AUG codons. Unexpectedly, the first AUG codon is required for efficient GCN4 expression under starvation conditions. This positive function appears to involve antagonism of the inhibitory effect of the 3' proximal AUG codons since it is dispensable in the absence of these sequences. The interaction between the upstream AUG codons is modulated by the trans-acting factors GCN2 and GCD1 in response to amino acid availability.

Primary structure of wild-type and mutant alleles of the PET494 gene of Saccharomyces cerevisiae.

The product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase. We have determined the DNA sequence of a 1.9 kb fragment carrying PET494. The sequence contains a single long open reading frame of 489 codons. This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon. The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5' ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame. The predicted PET494 protein has a highly basic amino-terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic. The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic. The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function. A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations linked to PET494.