to high cell densities and a well-defined, inexpensive media. Main disadvantages include significant glycosylation differences of secreted proteins comprised of high mannose, hyperglycosylatio consisting of much longer carboydrate chains than those found i higher eukaryotes, and the absence of secretory components for processing certain higher eukaryotic proteins(reviewed in Cregg, 1999). Because of these limitations, yeast systems will not be dis cussed in full detail in this chapter. More information on Pichia expression can be found in the following references: Higgins and Cregg(1998), Cregg, Vedvick, and Raschke (1993), and Sreekrishna et al. (1997) We all have our preferences for what are the best cell lines to se. Therefore, when setting up an expression laboratory, one should consider obtaining a variety of host cell lines. Listed are and reasons for their selection: CHO-DG-44 and Drosophila $2 (available from Invitrogen), based on consistency in growth, high level expression, and ability to be easily adapted to serum-free growth in suspension; COS for transient expression; HEK 293, versatile human cell line which can be used for both transient(but not as good as COS)and stable expression; and Sf9 a host cell for baculovirus infection, a system best suited for internalized pro- teins rather than secreted proteins. A majority of these cell lines can be grown in serum-free suspension culture, a property that facilitates ease of use and product purification as well as reducing cost Selecting an Appropriate Expression Vector e Once an appropriate host system has been chosen, it's time to d a suitable expression vector. For each of the host systems described above, there are a wide variety of vectors to choose A typical expression vector requires the following regulate elements necessary for expression of your gene: a promoter, trans- lational initiator codon, stop codon, a polyadenylation signal,a selectable marker, and several prokaryotic elements such as a bac terial antibiotic selection marker and an origin of replication for plasmid maintenance. (The presence of prokaryotic elements is for shuttling between mammalian and prokaryotic hosts. )There are numerous choices for each regulatory element, but unfortu nately there is no blueprint on which combinations will yield the highest expressing plasmid Trill et al
to high cell densities and a well-defined, inexpensive media. Main disadvantages include significant glycosylation differences of secreted proteins comprised of high mannose, hyperglycosylation consisting of much longer carboydrate chains than those found in higher eukaryotes, and the absence of secretory components for processing certain higher eukaryotic proteins (reviewed in Cregg, 1999). Because of these limitations, yeast systems will not be discussed in full detail in this chapter. More information on Pichia expression can be found in the following references: Higgins and Cregg (1998), Cregg, Vedvick, and Raschke (1993), and Sreekrishna et al. (1997). We all have our preferences for what are the best cell lines to use. Therefore, when setting up an expression laboratory, one should consider obtaining a variety of host cell lines. Listed are a few examples of cell lines that have been routinely used and reasons for their selection: CHO-DG-44 and Drosophila S2 (available from Invitrogen), based on consistency in growth, highlevel expression, and ability to be easily adapted to serum-free growth in suspension; COS for transient expression; HEK 293, a versatile human cell line which can be used for both transient (but not as good as COS) and stable expression; and Sf9 a host cell for baculovirus infection, a system best suited for internalized proteins rather than secreted proteins. A majority of these cell lines can be grown in serum-free suspension culture, a property that facilitates ease of use and product purification as well as reducing cost. Selecting an Appropriate Expression Vector Once an appropriate host system has been chosen, it’s time to find a suitable expression vector. For each of the host systems described above, there are a wide variety of vectors to choose from. A typical expression vector requires the following regulatory elements necessary for expression of your gene: a promoter, translational initiator codon, stop codon, a polyadenylation signal, a selectable marker, and several prokaryotic elements such as a bacterial antibiotic selection marker and an origin of replication for plasmid maintenance. (The presence of prokaryotic elements is for shuttling between mammalian and prokaryotic hosts.) There are numerous choices for each regulatory element, but unfortunately there is no blueprint on which combinations will yield the highest expressing plasmid. 506 Trill et al
Table 16.2 Promoter Strength Table Promoter Source rength EF-la Human elongation 40-160 Mizushima and Nagata(1990) factor 1a Human Boshart et al. (1985) cytomegalo immediate-early gene RSV Rous sarcoma Gorman et al. (1982) LTR SV40 late mian virus 40 1.1 Wenger, Moreau, and Nielsen ate gene SV40 early Simian virus 40 Early gene or Adenovirus major Mansour, Grodzicker, and late promoter rjan(1986) Beta-globin Mouse beta-globin 0.2 Hamer, Kaehler, and Leder promoter (1980) Beta-actin Human beta-actin ND Ng et al. ( 1985) Note: SV40 early promoter strength set as 1 for comparative purposes, and the numbers indicate how much stronger these Promoters Promoters are DNA sequences that recruit cellular factors and RNa polymerase to activate transcription of a particular gene They must contain a transcriptional start site, a CAat box, and TATA box. Examples of various mammalian promoters are giver in Table 16.2 The promoter strength is based on a compilation of compara tive experiments where various promoters were compared in tran sient experiments using the r1610 cell line(Thirion, Banville, and Noel, 1976). The strength of EF-la and CMV was derived from a comparison to the RsV LtR involving stable expression of arious monoclonal antibodies and tPA (Trill, 1998 unpublished) The EF-la promoter (available from Invitrogen) is by far the strongest promoter and a good choice if you want quick high-level expression. Polyadenylation Regions Polyadenylation occurs at a consensus sequence, AAUAAA and results in increased mRNA stability. Cleavage after the U by poly a polymerase adds a string of adenylate residues (Wahle and Keller, 1992). As with the promoters, there are a number of sources of polyadenylation regions. Several examples are shown in Table 16.3 Eukaryotic Expression 507
Promoters Promoters are DNA sequences that recruit cellular factors and RNA polymerase to activate transcription of a particular gene. They must contain a transcriptional start site, a CAAT box, and TATA box. Examples of various mammalian promoters are given in Table 16.2. The promoter strength is based on a compilation of comparative experiments where various promoters were compared in transient experiments using the R1610 cell line (Thirion, Banville, and Noel, 1976). The strength of EF-1a and CMV was derived from a comparison to the RSV LTR involving stable expression of various monoclonal antibodies and tPA (Trill, 1998 unpublished). The EF-1a promoter (available from Invitrogen) is by far the strongest promoter and a good choice if you want quick high-level expression. Polyadenylation Regions Polyadenylation occurs at a consensus sequence, AAUAAA, and results in increased mRNA stability. Cleavage after the U by poly A polymerase adds a string of adenylate residues (Wahle and Keller, 1992). As with the promoters, there are a number of sources of polyadenylation regions. Several examples are shown in Table 16.3. Eukaryotic Expression 507 Table 16.2 Promoter Strength Table Promoter Source Strength Reference EF-1a Human elongation 40–160 Mizushima and Nagata (1990) factor 1a CMV Human 4 Boshart et al. (1985) cytomegalovirus immediate-early gene RSV Rous sarcoma 2 Gorman et al. (1982) virus LTR SV40 late Simian virus 40 1.1 Wenger, Moreau, and Nielsen Late gene (1994) SV40 early Simian virus 40 1 Early gene Adeno major Adenovirus major 0.4 Mansour, Grodzicker, and late late promoter Tjian (1986) Beta-globin Mouse beta-globin 0.2 Hamer, Kaehler, and Leder promoter (1980) Beta-actin Human beta-actin ND Ng et al. (1985) promoter Note: SV40 early promoter strength set as 1 for comparative purposes, and the numbers indicate how much stronger these promoters are
Table 16.3 Polyadenylation Regions Poly a re Source Efficiency Bovine growth hormone SV40 late Simian virus 40 Herpes simplex virus thymidine kinase SV40 early Simian virus 40 Hep b Hepatitis B surface antigen Note: Sv40 l y(a)region strength set as 1 for comparative numbers indicate how much more efficient these polyadenylation regi The data above and polyadenylation regions are referenced in Pfarr et al. (1985 Drug Selection Markers Choice number three: What drug selection markers should one use? These genes provide resistance to a particular selective di and only cells in which the plasmid has been integrated will survive selection. Some effective choices are Blasticidin(Izumi et al., 1991), Histidinol, (Hartman and Mulligan, 1988), Hygromycin B(Gritz and Davies, 1983), Geneticin(G418)(Colbere-Garapin et al, 1981), Puromycin(de la Luna et al., 1988), mycophenolic acid( Mulligan and Berg, 1981), and ZeocinM(Mulsant et al 1988). Whatever marker you decide to use, remember, you will need to determine the effective concentration of drug for each cell line you use. Second, if you are on a tight budget, there is a huge disparity in cost of these drugs. Also there are environmental con cerns regarding waste disposal of the conditioned growth medium containing some of these drugs. Finally, if expression is unacceptably low, one amplify your gene copy number. Two such amplification systems are the use of dihydrofolate reductase (DHFR)as a drug selec- tion marker in the presence of methotrexate, a competitive inhibitor of DHFR(Kaufman, 1990) and inhibition of the enzyme glutamine synthetase (Gs) by methionine sulfoxide (MSX) (Bebbington et al., 1992) Amplification through the DHFR is by far the more popular of the two systems. DHFR version of folate to tetrahydrofolate, which is necessary in the synthesis of glycine, thymidine monophosphate, and the biosynthesis of purines. If the transfected plasmid contains a DHFR gene, use of he CHo DG-44 and dUK-Bll cell lines allows one to initially select cells in medium devoid of nucleotides and then to amplify gene copy number by selection with increasing concentrations of Trill et al
Drug Selection Markers Choice number three: What drug selection markers should one use? These genes provide resistance to a particular selective drug, and only cells in which the plasmid has been integrated will survive selection. Some effective choices are Blasticidin (Izumi et al., 1991), Histidinol, (Hartman and Mulligan, 1988), Hygromycin B (Gritz and Davies, 1983), Geneticin® (G418) (Colbere-Garapin et al., 1981), Puromycin (de la Luna et al., 1988), mycophenolic acid (Mulligan and Berg, 1981), and ZeocinTM (Mulsant et al., 1988). Whatever marker you decide to use, remember, you will need to determine the effective concentration of drug for each cell line you use. Second, if you are on a tight budget, there is a huge disparity in cost of these drugs. Also there are environmental concerns regarding waste disposal of the conditioned growth medium containing some of these drugs. Amplification Finally, if expression is unacceptably low, one solution is to amplify your gene copy number. Two such amplification systems are the use of dihydrofolate reductase (DHFR) as a drug selection marker in the presence of methotrexate, a competitive inhibitor of DHFR (Kaufman, 1990) and inhibition of the enzyme glutamine synthetase (GS) by methionine sulfoxide (MSX) (Bebbington et al., 1992). Amplification through the DHFR gene is by far the more popular of the two systems. DHFR catalyzes the conversion of folate to tetrahydrofolate, which is necessary in the synthesis of glycine, thymidine monophosphate, and the biosynthesis of purines. If the transfected plasmid contains a DHFR gene, use of the CHO DG-44 and DUK-B11 cell lines allows one to initially select cells in medium devoid of nucleotides and then to amplify gene copy number by selection with increasing concentrations of 508 Trill et al. Table 16.3 Polyadenylation Regions Poly A Region Source Efficiency BGH Bovine growth hormone 3 SV40 late Simian virus 40 2 TK Herpes simplex virus 1.5 thymidine kinase SV40 early Simian virus 40 1 Hep B Hepatitis B surface antigen 1 Note: SV40 early poly(A) region strength set as 1 for comparative purposes, and the numbers indicate how much more efficient these polyadenylation regions are. The data above and polyadenylation regions are referenced in Pfarr et al. (1985, 1986)