Improved Mammalian Expression Systems by Manipulating Transcriptional Termination Regions Dongjun Kim, † Jeong Do Kim, Kwanghee Baek, Yeup Yoon, ‡ and Jaeseung Yoon* Graduate School of Biotechnology, Kyung Hee University, Yongin City, Kyungki-Do 449-701, Korea Here we show that the transcriptional terminator element of human gastrin gene, which is the only element characterized to date in terms of its function in transcrip- tional termination, increases the transient expression levels of recombinant proteins. The expression of the -galactosidase gene was enhanced 3-4-fold in HeLa cells by inserting the terminator element of human gastrin gene at the 3′-side of the SV40 polyadenylation signal/cleavage site of the control vector (pSV--gal). This effect of the terminator element is orientation-dependent but not cell-specific since a similar enhancement of -galactosidase gene expression was detected in COS.M6 and CHO DG44 cells. The increased level of -galactosidase gene expression by the transcrip- tional terminator element of human gastrin gene turned out to arise from elevated cellular mRNA levels, suggesting that the terminator element stabilizes mRNA by enhancing proper 3′-end processing of mRNA. Introduction Expression systems using mammalian host cells are becoming increasingly important for the expression of genes cloned from higher eukaryotic cells. A large number of vectors are currently available, and most of these contain multiple elements, including a replicative origin, an efficient promoter element, mRNA processing signals, polylinkers, and selectable markers (1, 2). Enor- mous efforts have been made to improve mammalian expression systems mainly by improving promoter ele- ments and selectable markers (1, 2). One of the important components in the design of mammalian expression vectors is the element required for transcriptional termination. Continued transcription from an upstream promoter often interferes with down- stream promoter function (3, 4). The read-through of RNA polymerases at the 3′-end of the gene may result in the inefficient expression of a cloned gene. The transcription terminators in prokayotes have been well characterized and are widely used in microbial expression vectors (5). The 3′-end regions of eukaryotic genes are known to contain three important functional elements: a polyadenylation signal, a cleavage site, and a termina- tor element. Several polyadenylation signals and cleavage sites have been well characterized and widely used in mammalian expression vectors (1, 2). However, little information is available on the transcriptional terminator elements where RNA polymerases actually stop synthe- sizing mRNAs. Although a consensus sequence has been identified in the terminal region of nine eukaryotic genes (6), the only transcription terminator functionally identi- fied so far is the one existing at the 3′-end region of the human gastrin gene (7-9). Here we report that the application of the transcriptional terminator element of the human gastrin gene to a mammalian expression system enhances the transient expressions of the cloned genes. Materials and Methods Expression Plasmid Constructs. The 70 and 74 bp DNA containing the polyadenylation signal/cleavage site and the terminator element, respectively, of the human gastrin gene were prepared by synthesizing and anneal- ing the forward and reverse oligonucleotides (Figure 1A). pSV--gal (Promega Co.) containing a SV40 promoter/ enhancer, a -galactosidase reporter gene, and a SV40 polyadenylation signal/cleavage site was used as a basic vector for constructing various expression plasmids to monitor the effects of the terminator element of human gastrin gene (Figure 1B). The 74 bp terminator element of the human gastrin gene was inserted at the 3′-side of the SV40 polyadenylation signal/cleavage site of pSV-- gal vector in the forward and reverse orientations (pSV- SPA-GTF and pSV-SPA-GTR, respectively, in Figure 1B). The SV40 polyadenylation signal/cleavage site of pSV- -gal vector was replaced with the polyadenylation signal/ cleavage site of human gastrin gene, and the resulting plasmid was designated as pSV-GPA (Figure 1B). The terminator element of human gastrin gene was inserted at the 3′-side of the polyadenylation signal/cleavage site of pSV-GPA in the forward and reverse orientations (pSV-GPA-GTF and pSV-GPA-GTR, respectively, in Fig- ure 1B). The pSV2cat plasmid (ATCC), containing the chloramphenicol-acetyltransferase (CAT) reporter gene, was used to compensate for transfection efficiencies. Standard Techniques. Purification of expression plasmids, total RNA isolation, RT-PCR, and DNA blot hybridizations were performed using standard techniques (10). Cell Cultures and Transfection. HeLa, COS.M6, and CHO DG44 cells were grown in DMEM medium (GIBCO-BRL) supplemented with 10% Fetal Bovine Serum (GIBCO-BRL). Transient transfections of HeLa * To whom correspondence should be addressed. Phone: +8231- 201-2450. Fax: +8231-203-4969. E-mail: jsyoon@khu.ac.kr. † Current address: PanGen Biotech Inc. R&D Center, Kyungki Small Business Center, Suwon, Kyungki-Do 442-270, Korea. ‡ Current address: Mogam Biotechnology Research Institute, Yongin City, Kyungki-Do 449-910, Korea. 1620 Biotechnol. Prog. 2003, 19, 1620-1622 10.1021/bp0341186 CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 09/17/2003