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Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low‐density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 31:1172–1178, 2015

The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escierichia coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon and either the E. coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene. The expression of Met-bGH was low with either system. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation.High-level expression of a cloned gene in Escherichia coli generally involves the incorporation of the gene into a multicopy replicon, transcription of the gene from a strong promoter, and efficient translation of the mRNA (1-3). In this paper, we present our studies on the conditions necessary for high-level expression of bovine growth hormone (bGH) in E. coli. A cDNA clone for the full-length bovine growth pre-hormone was isolated by Miller et al. (4), and the coding region for mature bGH (5) was then cloned into pBR322 (unpublished data).Our initial approach to obtain high-level expression of bGH was to increase the gene dosage. This was achieved by using the thermoinducible runaway-replication plasmid pl-MIA (6). This plasmid was derived from pKN402 (7) and has a copy number of 10-15 per cell below 30'C and 1000-2000 per cell at 370C. pIMIA contains a kanamycin-resistance marker and the E. coli lipoprotein (lpp) promoter and ribosome binding site. When the bGH gene was cloned into pI-MIA, behind the lpp promoter, we observed that only small amounts of Met-bGH were synthesized. The poor expression of Met-bGH was due to the poor translational efficiency of the mRNA encoding Met-bGH, which was overcome by inserting additional codons 3' to the AUG initiation codon. To obtain high-level expression of Met-bGH, without the introduction of extra codons, we constructed a genetic system consisting of two contiguous cistrons. The first cistron contains the codons that enhanced the translational efficiency of Met-bGH, and the second cistron contains the Met-bGH coding sequence. We refer to this system as a two-cistron construction, and we propose that this approach will be generally applicable to improving gene expression in E. coli.MATERIALS AND ME...

A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A+T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.In recent years, many prokaryotic and eukaryotic genes have been expressed at high levels in Escherichia coli. The general approach has been to use a multicopy cloning vector with a strong promoter and an efficient ribosome binding site for the transcription and translation of the cloned gene (1, 2). However, the level of gene expression with these vectors varies widely for different eukaryotic genes. Low-level expression has been attributed to protein degradation by E. coli proteases (3) or to inefficient translation initiation of mRNAs containing heterologous gene sequences (4-8). Several studies suggested that the efficiency of translation initiation depends on the degree of complementarity between the Shine-Dalgarno (SD) sequence and the 16S rRNA, the distance between the SD sequence and the initiation codon, and the nucleotide sequence of this "window" region (9-16). There is evidence that the translational efficiency also depends on the sequence of the 5' untranslated region of the mRNA outside the SD sequence and the 5' end of the protein coding region (17)(18)(19).To reconcile these observations, it has been proposed that translation is inhibited when local secondary structur...

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