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Bacteria Mature Preproinsulin to Proinsulin

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Bacteria Mature Preproinsulin to Proinsulin Proc. Nati. Acad. Scd. USA Vol. 77, No. 7, pp. 3988992, July 1980 Biochemistry Bacteria mature preproinsulin to proinsulin (hybrid signal sequences/secretion/signal peptidase/immunoprecipitation/protein processing) KAREN TALMADGE, JIM KAUFMAN, AND WALTER GILBERT Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Contributed by Walter Gilbert, April 25, 1980 ABSTRACr By inserting the rat preproinsulin gene into the reus strain Cowan I was heat-killed and formalin-treated by bacterial prepenicillinase gene, we formed a variety of hybrid the method of Kessler (13) and resuspended (10% volume/ bacterial-eukaryotic signal sequences attached to proinsulin. Among these were the four following constructions: rat proin- volume) in NET buffer (50 mM Tris-HCI, pH 7.5/5 mM sulin attached to the entire penicillinase signal sequence and EDTA/0.15 M NaCi) (13). rat preproinsulin fused to all of, to hal of, or only to the first four Radiolabeling of Proteins. Escherlchla coil K-12 strains amino acids of the bacterial signal sequence. In all four cases, PR13 bearing insulin plasmids p287.47 (which produces protein «iore than 90% of the rat insulin antigen appeared in the peri- i27/+4), p241.1947 (protein i12/-21), p218.CB6 (protein plasmic space. By immunoprecipitation and determination of no insulin insert), the amino acid sequences ofthe radiolabeled products, we show i4/-21), or pKT41 (a control plasmid with that the bacteria correctly process both thebacterial and the and FMA10/kcI&s7 bearing insulin plasmid p280.1947 (protein eukaryotic signal sequences of these hybrid proteins. The i25/-21) [all described by Talmadge et al. (7)] were grown cleavage of the eukaryotic signal by bacterial peptidase, in this overnight in 2YT medium (14) supplemented with thymidine case, generates proinsulin. at 40 Atg/ml for FMA10. Fifty microliters was inoculated into 10 ml of S medium (15) supplemented either with thiamine at Secretion is an essential feature of cells. The precursors of almost 10 Mg/mi and thymidine at 40 Mg/mi for FMA10 or with L- all secreted proteins, both eukaryotic and prokaryotic, contain leucine and L-threonine at 40 Mug/ml each for PRiS, and then an amino-terminal extension (ref. 1; see ref. 2 for review). The grown to ODsso of 0.3. Five millicuries of H235SO4 was added signal hypothesis (1, 3) proposes that this peptide, the signal to all cells (except PR13/p287.47) and incubation was continued sequence, serves to bind the protein to the membrane and then 1 hr with shaking at 370C (PR13) or 34WC (FMA10). PR13 to lead it across. Sometime during transport, the signal sequence bearing p287.47 was harvested, resuspended in 10 ml of S is removed and the preprotein is thereby processed to the ma- medium supplemented with L-threonine at 40 Mg/mI and in- ture form. cubated for 1 hr at 370C with shaking with 5 mCi of H2aWSO4 In bacteria, direct evidence establishes that the signal se- and 2.5 mCi of [3H]leucine. quence is essential for transport. Mutations have been described Immunoprecipitations. Labeled cells were harvested, for two proteins (4, 5) that lead to the accumulation of the resuspended in 100 Ml of Tris-HCI, pH 8/20% sucrose and in- mutant product in the cytoplasm as the preprotein. In each case, cubated 15 min with 100 Ml of lysozyme at 20mg/ml in 20 mM the mutation results in an amino acid replacement in the signal for 5 sequence. Furthermore, rat proinsulin, attached to a complete EDTA, pH 8. The cells were pelleted by centrifugation bacterial signal sequence, is efficiently transported (6, 7); min at 10,000 rpm in a Sorvall SS-34 rotor, and the supernatant lacking a signal, it is not (7). was diluted with 800 Ml of 150 mM Tris-HCI, pH 8/2% Triton The mechanism of secretion is quite general. Shields and X-100/0.2 M EDTA. Alternately, labeled cells were harvested, Blobel (8) have used dog pancreas microsomes to segregate and resuspended in 100 Ml of the Tris/sucrose buffer as above, in- process fish preproinsulin. Moreover, Fraser and Bruce (9) cubated with 100 Ml of lysozyme in EDTA as above, and lysed showed that chicken ovalbumin is secreted (50%) from bacterial with 800 Ml of Triton buffer as above. The cell debris was pel- cells when that gene is cloned in bacteria. Ovalbumin is unique leted at 16,500 rpm for 1 hr in a Sorvall SA600 rotor. A 200- to among secreted proteins studied so far: it does not have an 1000-fold excess [as determined by radioimmunoassay (7)] of amino-terminal extension (10), although it may contain an in- an IgG fraction of guinea pig anti-insulin serum was added to ternal signal sequence (11). We have recently shown (7) that each supernatant, and the mixture was held for 1 hr at 370C and a normal eukaryotic signal sequence, the rat preproinsulin signal then 1 hr on ice. One hundred microliters of heat-killed, for- sequence, directs the efficient secretion of rat insulin antigen malin-treated S. aureus (10% vol/vol) was added, and the in bacteria. Is this eukaryotic presequence processed? mixture was incubated for 30 min on ice and washed by the method of Kessler (13). MATERIALS AND METHODS Polyacrylamide Gel Electrophoresis. S. aureus bacteria complexed to proteins to be analyzed by polyacrylamide gel Materials. Chicken lysozyme, chicken ovalbumin, sperm electrophoresis were resuspended in 100 Ml of sample buffer whale myoglobin, and iodoacetamide were from Signma; bovine [200 mM Tris.HCl, pH 6.8/10% (vol/vol) glycerol/0.01% proinsulin was a gift of Donald Steiner; human f32-microglo- (wt/vol) bromophenol blue/5 mM EDTA/2% NaDodSO4/ bulin was a gift of Cox Terhorst; H2asSO4 (carrier-free) and dithiothreitol (freshly added to 10 mM)], boiled 3 min, allowed L-[4,5-3H(N)]leucine (50 Ci/mmol; 1 Ci = 3.7 X 1010 bec- to cool to room temperature, and incubated for 20 min with 20 querels) was purchased from New England Nuclear. An IgG Ml of 0.5 M iodoacetamide. Thirty microliters of sample buffer fraction of anti-insulin antiserum (from Miles) was prepared made 200 mM in dithiothreitol was added, the room temper- as described by Broome and Gilbert (12). Staphylococcus au- ature incubation was continued another 10 min, and the bac- teria were removed by centrifugation. Aliquots (10-50 Ml) were The publication costs of this article were defrayed in part by page loaded onto a 15% Laemmli gel charge payment. This article must therefore be hereby marked "ad- NaDodSO4/polyacrylamide vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: HPLC, high-performance liquid chromatography. 3988 Downloaded by guest on September 25, 2021 Biochemistry: Talmadge et al. Proc. Natl. Acad. Sc. USA 77 (1980) 3989 (16) with 7 M urea in the bottom gel and 2 mM EDTA added prepenicillinase wild-type amino acid before the amino acids to all buffers. One microgram each of sperm whale myoglobin, encoded by the insertion of the Pst restriction site, the second chicken lysozyme, human 132-microglobulin, and bovine referring to the first amino acid of preproinsulin (negative proinsulin were run as molecular weight markers. The stained numbers) or proinsulin (positive numbers). Either the complete dried gel was autoradiographed on Kodak XR-5 film. S. aureus bacterial signal sequence or the major part of the eukaryotic bacteria complexed to proteins whose sequences were to be signal served to transport efficiently to the periplasm. determined were resuspended in 100 Ad of Maizel gel buffer Immunoprecipitation and Polyacrylamide Gel Electro- (17), boiled 3 min, and centrifuged 5 min in a Sorvall SS-34 rotor phoresis. We grew cells containing the insulin gene plasmids at 10,000 rpm. The supernatant was run on a 15% Maizel gel in a low-sulfate medium and labeled the proteins with H23-SO4 (17), the wet gel was autoradiographed on Kodak XR-5 film for or with both H2asSO4 and [3H]leucine. We isolated the labeled 1 hr at 4VC, and the protein was eluted from a crushed gel slice protein products from the periplasmic fraction by adding an for 8 hr at room temperature with shaking in 1-2 ml of 50 mM excess of anti-insulin IgG and immunoprecipitating by incu- ammonium bicarbonate, pH 7.5/0.2 mg of ovalbumin per bating with formalin-treated, heat-killed S. aureus (13). Fig. ml/0.2 mM dithiothreitol/0.1% NaDodSO4. The crushed gel 2 shows an autoradiogram of the electrophoresis of the immu- was removed by filtration through silicone-treated glass wool noprecipitated proteins on a Laemmli NaDodSO4/polyacryl- and the protein was lyophilized. amide gel (16) containing urea and EDTA. A dark new band Amino Acid Sequence Analysis. The protein isolated from appears in the four samples from insulin-antigen-producing a Maizel gel was resuspended in 100 Ad of distilled water and cells (Fig. 2, lanes a-d) that is absent in the control precipitation 3 mg of ovalbumin was added. The proteins were precipitated (Fig. 2, lane e). Without processing, i25/-21 would have 142 in 5 vol of acetone and resuspended in 200,ul of 70% (wt/vol) amino acids, i12/-21 would have 130, i27/+4 would have 121, formic acid, and then 3 mg of Polybrene (Aldrich) in 200 ,l of and i4/-21 would have 118. Instead, i27/+4 (Fig. 2, lane a) 70% formic acid was added. Between 20,000 and 300,000 cpm is larger than the other three, which are all the same size and was loaded onto a Beckman sequenator, updated model 890B, run close to, but slower than, the bovine proinsulin standard; and successive steps of Edman degradation were performed, bovine proinsulin is 5 amino acids shorter than rat proinsulin using a 0.1 M Quadrol program (18).
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