Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells

Comp. Biochem. Physiol. Vol. 93B, No. 4, pp. 803-806, 1989 Printed in Great Britain

0305-0491/89 $3.00 + 0.00 © 1989 PergamonPress plc

CELL-FREE TRANSLATION IN LYSATES FROM S P O D O P T E R A FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE) CELLS MAVIS R. SWERDEL and ANN MARIE FALLON Department of Entomology, University of Minnesota, 1980 Folwell Ave, St Paul, MN 55108, USA

(Received 6 December 1988) Abstract--1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.

INTRODUCTION

Cell-free translation systems have provided an important tool for analysis of the interaction of m R N A with the various factors involved in protein synthesis, for determining the coding capacity of purified viral and cellular mRNAs, and for investigating the posttranslational modification of proteins. The earliest approaches to developing cell-free translation systems from insect sources included analysis of protein synthetic activity in intact organisms as well as in homogenates and subcellular fractions from various species including the silkmoth Bombyx mori (Takeyama et al., 1958; Faulkner and Bheemeswar, 1960; Suzuka et al., 1962), Drosophila (Fox et al., 1965; Rose and Hilman, 1969), the housefly Musca domestica (Litvak et al., 1967; GadaUah et al., 1971), the beetle Tenebrio molitor (Ilan, 1968; Lassam et al., 1975), the cricket Acheta domesticus (Kaulenas, 1970), and the blowflies Calliphora erythrocephala (Sekeri et al., 1968) and Lucilia cuprina (Williams and Birt, 1972). Although these early studies demonstrated that the potential difficulties in developing translation systems from insect sources were not insurmountable, most of these systems have not enjoyed further development using more current molecular approaches. More recently, several independent investigators have developed translation systems from insect cells in culture. Davis and Hartig (1977) have described requirements for cell-free protein synthesis in extracts from a cell line derived from the codling moth, Laspeyresia pomonella. Drosophila melanogaster lysates have been used extensively for analysis of heat-shock induced translational control of protein synthesis (Scott and Pardue, 1981; Storti et aL, 1980; Sanders et al., 1986) and for studies on the translation of mRNAs from black beetle virus, a bipartite R N A virus in the family Nodaviridae that replicates in Drosophila cells (Guarino et al., 1981; Friesen and Rueckert, 1984). A cell-free translation system has

also been developed from cultured Aedes albopictus (mosquito) cells (Gillies and Stollar, 1981). Lysates from mosquito cells were stable to freezing and were supplemented with a purified ribonuclease inhibitor, which replaced the rat liver supernatant fraction used in some Drosophila lysates (Scott and Pardue, 1981). The Aedes system has been used to analyze the effects of viral infection on protein synthesis in mosquito cells (Gillies and Stollar, 1982) and also to differentiate between membrane and cytoplasmic changes in cells resistant to antibiotics that interact with the ribosome (Fallon and Stollar, 1982a,b). We have recently found t h a t the commercially available components used to supplement wheat germ translation kits can be substituted for the reaction components described by Gillies and Stollar (1981) for mosquito cell lysates (unpublished data). This observation led us to consider the possibility that translation systems representing a wide variety of sources could be based on relatively standard reaction conditionsl The importance of cell lines from Spodoptera frugiperda for investigating baculovirus replication led us to test these conditions with lepidopteran cells. MATERIALS AND METHODS

Preparation of extracts Spodopterafrugiperda cells (line IPLB-SF) were obtained at the 656th passage from Dr J. Maruniak (U. Florida, GainesviUe) and maintained in Grace's medium (GIBCO) supplemented with heat inactivated fetal bovine serum at a final concentration of 10%. For preparation of lysates, cells in exponential growth were pooled from 10-15 plates (100 mm), collected by centrifugation, and washed twice in phosphate-buffered saline (Dulbecco and Vogt, 1954) lacking calcium and magnesium. Packed cells were resuspended in 1.5 volumes of lysis buffer [20mM Hepes, pH %6, containing 10 mM potassium acetate and 5 mM dithiothreitol (DTT)]. After 10min on ice, the swollen cells were broken in a glass homogenizer using a Teflon pestle (30 strokes). The homogenate was mixed with one-ninth volume

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of 10 × buffer (100 mM Hepes, pH 7.6, 1.5 M potassium acetate, 20 mM magnesium acetate, and 50 mM DTT) using 5 additional strokes with the homogenizer. The extract was clarified by low-speed centrifugation (2500 rpm for 10 min), and the supernatant was centrifuged at 10,000g for 20 rain. The resulting supernatant was collected, care being taken to avoid the lipid layer, and stored in aliquots at - 7 0 C . Lysates were stable for 2 freeze thaw cycles; after a third, activity was reduced by 50%. Frozen lysates were stable for several months at - 7 0 C .

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Conditions Jor in vitro translation The reaction contained the following components at the indicated final concentrations: creatine phosphokinase (100#g/ml), creatine phosphate (6raM), Hepes buffer, pH7.6 (25mM), GTP (0.5mM), spermidine (126#M), potassium acetate (100mM), magnesium acetate (1 mM), DTT (4mM), human placental ribonuclease inhibitor (6 units; Bethesda Research Laboratories), and [35S]methionine (5 pCi; 1000--1150Ci/mmol; New England Nuclear). The reaction mixture was held on ice, and translation was initiated by the addition of lysate (10 pl) to give a final reaction volume of 20 y1. Reactions were incubated at 28°C for 1 hr, and activity was measured by determining acid-precipitable radioactivity on filter papers as described by Mans and Novelli (1961). Background values represent acid-precipitable radioactivity in samples taken immediately after addition of lysate.

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Fig. 1. Incorporation of [35S]methionine into acid-precipitable material as a function of time. Extracts were incubated for the indicated time under the conditions described in Materials and Methods. Background values (3821 cpm) have been subtracted; the 1 hr point represents a 17,5-fold stimulation of incorporation above background levels.

ence of endogenous mRNA templates represents both the elongation of previously initiated polypeptides as well as de novo initiation of new polypetide chains. Translation of purified exogenous mRNA, however, is specifically dependent upon the ability of RESULTS the lysate to carry out initiation of new polypeptide chains. To test whether initiation was occurring in Preparation of cell-free extracts this system, we treated the lysate with pactamycin Lysates were prepared from S. frugiperda cells (Table 1), which at concentrations below 10/IM according to the protocol described earlier for preferentially inhibits initiation of protein synthesis mosquito cells (Gillies and Stollar, 1981) with two (Lodish et al., 1971). At 5 ~M pactamycin, only 16% exceptions. The 6 hr incubation of cells in serum-free of the initial level of [35S]methionine incorporation medium prior to preparation of the extract was was measured, and at 50 #M, protein synthesis was omitted, and purified human placental ribonuclease almost completely inhibited (see also Brown et al., inhibitor was added at the time of assay, rather than 1983). Cycloheximide, which inhibits both initiation during preparation of the lysate. With mosquito cells, and polypeptide chain elongation (Olenick, 1977), these precautions were thought to minimize ribo- also inhibited translation in Spodoptera lysates nuclease activity in the resulting extracts. It seems (Table 1), giving approximately 50% inhibition of possible that lepidopteran cells may contain lower protein synthesis at 0.54 mM. Similarly, in cell-free levels of endogenous ribonuclease than do dipteran extracts from the codling moth, 0.21 mM cyclocells, but direct investigation of degradative activity heximide was required for inhibition of amino acid in the lepidopteran extracts has not been undertaken. incorporation by about 50% (Davis and Hartig, 1977). In contrast, only 25 ~M cycloheximide was Cell-free translation required for 50% inhibition of protein synthesis in Translation of endogenous mRNA in extracts from lysates from mosquito cells (Fallon and Stollar, Spodoptera cells increased with time for 45-60 min, 1982a). Translation in cell-free extracts generally shows a when net incorporation of [35S]methionine into acidprecipitable material was typically 15 to 20-fold marked sensitivity to magnesium and potassium conhigher than the background incorporation measured centrations. For translation of endogenous mRNA in immediately after addition of extract to the reaction Spodoptera lysates, the optimal concentration of mix (Fig. 1). It should be noted that the lysate was magnesium was approximately 1 mM; that for potassupplemented with an energy-generating system sium was 100 mM. It should be noted, however, that (creatine kinase/creatine phosphate) and GTP, but optimal salt concentrations in translation mixtures may co-vary (Brown et al., 1983), and conditions for exogenous amino acids other than [35S]methionine were not included in the reaction mixture. On SDS- translation of total endogenous mRNA may need to polyacrylamide gels, the [35S]methionine-labelled be adjusted for maximal translation of specific translation products made by Spodoptera lysates purified mRNAs. In lysates from some sources, varied in mass from greater than 45 to less than polyamines have been shown to stimulate trans6 kDa, suggesting that the endogenous mRNA con- lational activity by several-fold. In our hands, howsisted of numerous transcripts that varied consider- ever, spermidine gave at best a small (25%) increase in activity at concentrations between 110 and 150 pM ably in size (Fig. 2, lane 1; see also Scott and Pardue, (data not shown). 1981). To test whether the conditions described in the In cell-free systems, incorporation of amino acid precursor into acid-precipitable material in the pres- Materials and Methods allow for correct initiation of

Insect cell-free translation

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Table 1. Effect of protein synthesisinhibitors Pactamycin Proteinsynthesis Cyeloheximide Protein synthesis (uM) (% of Control) (mM) (% of Control) 0 100 0 100 0.1 83 0.18 80 0.5 40 0.54 52 1.0 27 1.1 39 5.0 16 --10 15 --50 9 --Incorporation of [35S]methioninewas measured after 60 min in assays containing the indicated amounts of pactamycin or cyeloheximide. Values are averaged from two independent experiments. exception of a prominent band at 16,000 daltons, suggesting that correct initiation of globin m R N A had occurred, and full length translation product was made (Fig. 2, lane 2). DISCUSSION

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Fig. 2. Synthesis of globin by micrococcal nuclease-treated lysate. Spodopterafrugiperda lysate was treated with micrococcal nuclease at a final concentration of 20 #g/ml in the presence of I mM CaC12. After I hr on ice, the nuclease was inactivated by addition of EGTA to a final concentration of 2 mM. Lane I shows incorporation of [35S]methionine into protein from endogenous mRNA in untreated lysate. Lane 2 shows synthesis of a 16 kDa translation product from a portion of the same lysate after treatment with micrococcal nuclease and supplementation with globin mRNA (0.5/ag in a 40pl reaction volume). Acid-precipitable radioactivity measured 18 x 103cpm/pl for the untreated lysate and 5.5 x 103 cpm/#l for the globin-primed reaction. Translation products were analyzed on a 12.5% SDS-polyacrylamide gel; an autoradiogram (6 day exposure) is shown. No translation products were detected in control samples containing micrococcal nuclease-treated lysate without exogenous globin mRNA (data not shown). translation from an exogenous m R N A , extract was treated with micrococcal nuclease according to the procedure described by Gillies and Stollar (1981) to reduce the a m o u n t of endogenous m R N A , and the treated extract was supplemented with purified globin m R N A . Analysis of the translation products on an SDS gel showed a substantially reduced incorporation of [35S]methionine into labelled polypeptides in the treated, globin-supplemented extract, with the

In spite of the increasing importance of cell lines from Spodopterafrugiperda and other lepidoptera for production of bioengineered products using baculovirus expression vectors (Luckow and Summers, 1988), few attempts have been made to extend to these cells technologies that will ultimately be useful for exploiting their full potential. We have shown that lysates from Spodoptera cells can be used for in vitro protein synthesis under conditions similar to those described earlier for mosquito cells. Optimal translation of endogenous m R N A required magnesium (1 mM) and potassium (100mM), but amino acids in excess of those present in the cell extract were not required. The Spodoptera translation system is substantially simpler than that described earlier for cells derived from the codling moth, Laspeyresia pomonella (Davis and Hartig, 1977) in that extensive preparation of subcelluar fractions is not required. Both of these lepidopteran cell-free systems provide linear incorporation of labelled amino acid into acid-precipitable material for approximately 1 hr, and show the expected inhibition by agents that act at the level of protein synthesis initiation and elongation. Lysates from cultured mosquito cells have been used successfully to study glycosylation of viral proteins (see for example, Gillies and Stollar, 1981). Although the importance of posttranslational modification of bioengineered proteins has long been recognized, glycosylation of proteins produced by baculovirus vectors has been investigated only indirectly (reviewed by Luckow and Summers, 1988). We anticipate that further refinement of in vitro translation systems, coupled with direct investigation of enzyme activities in cell extracts, will provide additional insight into the processing of foreign proteins in lepidopteran cells.

Acknowledgements--This work was supported by Grant AI 20385 from the National Institutes at Health, by Grant SO7 RR05908 administered by the NIH Division of Research Resources through the University of Medicine and Dentistry of N.J. School of Osteopathic Medicine, and by the University of Minnesota Agricultural Experiment Station. This is contribution 16,538 from the University of Minnesota Experiment Station, St Paul, MN. We thank Letha Fields for typing the manuscript, and Dr T. J. Kurtti for reading the manuscript.

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