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The signal sequence is composed of three domains: N, H, and C. The N domains of signal sequences are shown in bold, whereas the C domains are underlined.

2. Materials

1. E. coli XL1-Blue, BL21(DE3), HB101, and MC4100. Competent cells can be prepared using the standard calcium chloride method.

2. Recombinant plasmids: pUCOb (6), pTrcSOb4 (7), pTrcSObD (7), pTHKCSFmII (8), and pJS101AP (9; see Fig. 2 and Note 1).

3. Chromosomal DNA of E. coli W3110.

4. Luria-Bertani (LB) medium and LB agar plates: sterilized by autoclaving.

5. Ampicillin: used for both liquid and agar cultivation at a concentration of 50 |J,g/mL (Sigma, St. Louis, MO).

7. Isopropyl-ß-D-thiogalactopyranoside (IPTG) (Sigma).

12. H2O: sterilize before use.

13. Polymerase chain reaction (PCR) purification kit (Qiagen, Chatsworth, CA).

14. Expand High Fidelity PCR system (Roche, Mannheim, Germany).

15. Agarose (FMC Bioproducts, Rockland, ME).

16. Sterile toothpick.

17. Restriction enzymes (New England Biolabs [NEB], Beverly, MA).

19. QIAquick gel extraction kit (Qiagen).

20. Ni-nitrilotriacetic acid (Ni-NTA) superflow column (Qiagen).

22. Ex-Taq DNA polymerase (TaKaRa, Tokyo, Japan).

23. Dialysis membrane (molecular weight cut-off [MWCO] of 3500, Spectrum Lab Inc., Laguna Hills, CA).

endoxylanase signal sequence endoxylanase

BomHI^

lad pJSlOlAP

Xbal transcription terminator lad pJSlOlAP

B endoxylanase signal sequence obese gene

lad 11 pTrcSOb4

Xba\ transcription terminator lad 11 pTrcSOb4

£ endoxylanase signal sequence hG-CSF gene

Bamm' lad pTHKCSFmll transcription terminator

Fig. 2. Structures of plasmids: (A) pJS101AP, (B) pTrcSOb4, and (C) pTHKCSFmII.

3. Methods

3.1. Construction of Secretion Vector of Human Leptin

3.1.1. Vector Preparation

1. Add the following to a sterile Eppendorf tube: 10 pL of a 100 ng/pL solution of pJS101AP, 2 pL 10X BamHI NEB buffer, 8 pL H2O, and 0.4 pL BamHI and PstI (NEB).

2. Mix the contents, and incubate the solution at 37°C for 2 h.

3. Separate the digested gene product by electrophoresis on a 0.7% (w/v) agarose gel.

4. Purify the DNA fragments using a gel extraction kit.

3.1.2. Preparation of Insert DNA

1. Amplify the human obese gene (see Note 2) using pUCOb (6) as a template for the PCR reaction with 50 pL that contains 5 pL 10X PCR reaction buffer, 2 pL each of 20 pM primer (see Note 3), 1 pL of 0.2 ng/pL template, and 1.25 U Ex-Taq DNA polymerase (see Note 4).

2. Purify the PCR product with the PCR purification kit following the manufacturer's instructions.

3. Partially digest the purified PCR product with the PstI restriction enzyme and fully digest with BamHI restriction enzyme.

4. Separate the digested gene product by electrophoresis on a 0.7% agarose gel.

5. Purify the DNA fragments using a gel extraction kit.

3.1.3. Construction of Human Leptin Secretion Vector

1. Ligate digested DNA vector and PCR product (obese gene) by adding 100 ng vector and 300 ng PCR product into the 20 pL ligation mixture (T4 DNA ligase and buffer).

3. Transform E. coli XL1-Blue competent cells (200 pL) with 5 pL ligation mixture.

4. Dilute with 800 pL LB medium without the antibiotic.

6. Spread the cells on LB plates with ampicillin (or appropriate antibiotics if different antibiotic markers are used).

7. Incubate the plates overnight at 37°C.

8. Individual colonies are screened for the correct clone by miniplasmid preparation, then by restriction enzyme digestion.

9. Verify the correct sequence of the obese gene in the constructed vector (pTrcSOb4; see Fig. 2B) by DNA sequencing (see Note 5; 7).

3.2. Protein Expression and Periplasmic Fractionation

3.2.1. Protein Expression

1. Transformation of E. coli HB101 with the constructed vector pTrcSOb4 (see Note 6).

2. Inoculate E. coli HB101 (pTrcSOb4) into the LB medium that contains ampicillin.

3. Cultivate the cells to an optical density (OD600) of approx 0.6 with shaking (150 rpm) at 37°C.

4. Add IPTG to a final concentration of 1 mM for gene expression (see Note 7).

5. Cultivate the cells for an additional 5 h.

Fig. 3. SDS-PAGE analysis of periplasmic fractionation from E. coli BL21(DE3) harboring pTrcKObD. Lane 1, molecular mass standard; lane 2, E. coli BL21(DE3; no plasmid); lane 3, total proteins; lane 4, periplasmic proteins; lane 5, insoluble proteins; lane 6, soluble proteins. (Figure reproduced with permission from ref. 7.)

Fig. 3. SDS-PAGE analysis of periplasmic fractionation from E. coli BL21(DE3) harboring pTrcKObD. Lane 1, molecular mass standard; lane 2, E. coli BL21(DE3; no plasmid); lane 3, total proteins; lane 4, periplasmic proteins; lane 5, insoluble proteins; lane 6, soluble proteins. (Figure reproduced with permission from ref. 7.)

3.2.2. Periplasmic Fractionation

1. Centrifuge 1 mL induced culture sample in a microcentrifuge for 10 min at 7000g and 4°C.

2. Discard the supernatant (see Note 8).

3. Resuspend the cell pellet thoroughly in 0.4 culture volumes of 30 mM Tris-HCl, pH 8.0, plus 20% sucrose.

4. Add EDTA to a final concentration of 2 mM (see Note 9).

5. Incubate for 10 min at room temperature with shaking.

6. Collect the cells by centrifugation for 10 min at 10,000g and 4°C.

7. Remove all of the supernatant as far as possible.

8. Resuspend the pellet in the same volume of ice-cold 5 mM MgSO4 (see Note 10).

9. Stir the cell suspension slowly for 15 min on ice (see Note 11).

10. Centrifuge for 10 min at 10,000g and 4°C.

11. Transfer sample from the supernatant to a microcentrifuge tube (periplasmic fraction).

12. Resuspend the pellet in the same volume of TE buffer for preparing the cytoplasmic fraction (see Note 12).

13. Store at -20°C until sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis or activity assay (see Note 13 and Fig. 3).

3.2.3. Soluble and Insoluble (Inclusion Body) Fractionation

1. Centrifuge 1 mL induced culture sample in a microcentrifuge for 10 min at 7000g and 4°C.

2. Discard the supernatant.

3. Resuspend the cell pellet thoroughly in 0.2 mL TE buffer, pH 8.0.

4. Disrupt the cells by sonication using Sonics Vibra Cell (Branson Ultrasonic Co., Danbury, CT) for 1 min at 40% output.

5. Centrifugation for 10 min at 10,000g and 4°C.

6. Transfer sample from the supernatant to a microcentrifuge tube (soluble fraction).

7. Resuspend the pellet in the same volume of TE buffer, pH 8.0, to prepare the insoluble fraction.

8. Store at -20°C until SDS-PAGE analysis.

3.3. Enhancing the Efficiency of Secretory Production of Human Leptin

3.3.1. Vector Construction for DsbA Coexpression

1. Amplify the dsbA gene (7) by PCR using E. coli W3110 chromosome as a template. The PCR reaction is carried out in 50 \\L solution that contains 5 \\L 10X PCR reaction buffer, 2 \L each of 20 \M primer, 1 \L of 0.2 ng/\L template, and 1.25 U Ex-Taq DNA polymerase (see Note 14).

2. Purify the PCR product with the PCR purification kit following manufacturing instructions.

3. Digest pTrcSOb4 and the purified PCR product with Ndel restriction enzyme.

4. Separate the digested gene product by electrophoresis on 0.7% (w/v) agarose gel.

5. Purify the DNA fragments using a gel extraction kit.

6. Ligate digested DNA vector (100 ng) and PCR product (300 ng) by using T4 DNA ligase.

7. Verify the correct sequence of the insert DNA in the constructed vector (pTrcSObD) by DNA sequencing (7; see Note 15).

3.3.2. Protein Expression and Fractionation

1. Transformation of E. coli BL21(DE3) with the constructed vector pTrcSObD.

2. Cultivate the cells to an OD600 of approx 0.6 with shaking at 37°C.

3. Add IPTG at a final concentration of 1 mM for gene expression.

4. Cultivate for another 5 h.

5. Fractionation of induced cells for the SDS-PAGE analysis or purification as described earlier (see Fig. 4).

3.4. Expression of hG-CSF in E. coli

3.4.1. Expression of hG-CSF in E. coli

1. Transformation of E. coli MC4100 with the plasmid pTHKCSFmll (8; see Note 16).

2. Cultivate the cells to an OD600 of approx 0.6 with shaking at 37°C.

3. Add IPTG at a final concentration of 1 mM for gene expression.

4. Cultivate for an additional 5 h.

5. Fractionation of induced cells for the SDS-PAGE analysis or purification as described earlier.

3.4.2. Purification of Secreted hG-CSF

1. Centrifuge 50 mL induced culture for 10 min at 7000g and 4°C, and discard the supernatant.

2. Resuspend the cell pellet in 50 mL resuspension buffer: 50 mM NaH2PO4, 0.3 M NaCl, and 10 mM imidazole, pH 8.0.

3. Disruption by sonication with Sonics Vibra Cell for 20 min at 40% output.

4. Centrifugation for 30 min at 14,000g and 4°C.

Fig. 4. SDS-PAGE analysis of reduced and nonreduced inclusion body of human leptin produced in E. coli BL21(DE3) harboring pTrcKOb4 and pTrcKObD. Lane 1, molecular mass standard; lanes 2 and 3, E. coli BL21(DE3) (pTrcKOb4); lanes 4 and 5, E. coli BL21(DE3) (pTrcKObD); (+) with dithiothreitol (DTT); (-) without DTT; reduced leptin; (>) oxidized leptin; (►) intermolecular disulfide-linked inclusion bodies. (Figure reproduced with permission from ref. 7.)

Fig. 4. SDS-PAGE analysis of reduced and nonreduced inclusion body of human leptin produced in E. coli BL21(DE3) harboring pTrcKOb4 and pTrcKObD. Lane 1, molecular mass standard; lanes 2 and 3, E. coli BL21(DE3) (pTrcKOb4); lanes 4 and 5, E. coli BL21(DE3) (pTrcKObD); (+) with dithiothreitol (DTT); (-) without DTT; reduced leptin; (>) oxidized leptin; (►) intermolecular disulfide-linked inclusion bodies. (Figure reproduced with permission from ref. 7.)

(kDa)

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