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Apr 2019

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A PhoA-STII Based Method for Efficient Extracellular Secretion and Purification of Fab from Escherichia coli
一种基于phoa stii技术的 大肠杆菌中高效分泌表达的fab片段纯化   

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Abstract

In comparison with full-length IgGs, antigen binding fragments (Fabs) are smaller in size and do not require the complexed post-translational modification. Therefore, Fab can be cost-effectively produced using an Escherichia coli (E. coli) expression system. However, the disulfide-bonds containing exogenous protein, including Fab, tend to form insoluble inclusion bodies in E. coli, which has been the bottleneck for exogenous protein expressions using this system. The secretory expression of proteins in periplasm or extracellular medium are promising strategies to prevent the formation of inclusion bodies to improve the efficiency to produce Fabs from E. coli. The extracellular expression is of particularly interest since it releases the product into the medium, while periplasmic expression yield is limited to the periplasm space. In addition, the extracellular expression allows for the direct harvesting of proteins from the culture supernatant, sparing the procedures of cell lysis and reducing contamination of host cell protein or DNA. Using anti-VEGF Fab as an example, here we provide a protocol based on the alkaline phosphatase (phoA) promoter and the heat-stable enterotoxin II (STII) leader sequence. Using phosphate starvation to induce the secretory expression, the protocol could be generally used for the efficient production of Fabs.

Keywords: Fab (Fab), PhoA (PhoA), STII (STII), Extracellular production (细胞外合成), Secretion (分泌)

Background

Due to its clear genetic background, easy manipulation, and cost-effective production, E. coli is widely employed for exogenous gene expression, especially those of lower molecular weight and simpler conformational structures (Gupta and Shukla, 2017). The expression of foreign proteins in E. coli is mainly divided into three categories, inclusion bodies expression, intracellular soluble expression in periplasmic space, and extracellular secretion into medium (Jalalirad, 2013; Gupta and Shukla, 2017; Zhou et al., 2018). At present, most of mammalian-sourced foreign proteins are expressed in E. coli intracellular region, either in the form of inclusion bodies or soluble. But both forms have their respective disadvantages for subsequent processes. The inclusion bodies need to be tediously denatured and renatured to recover the target proteins with correct refolded structures, and the processes tend to cause reduction in the biological activity and yield (Panda et al., 2003; Nelson and Reichert, 2009). The periplasmic space of E. coli can provide an oxidative environment conducive to the formation of disulfide bonds, but the yield is usually limited by the capacity of the periplasmic space as well as the leading capability of signal peptides (Lobstein et al., 2012; Ellis et al., 2017). Compared with the two processes above, extracellular protein expression is not restricted to the intracellular space, allows convenient enrichment of target proteins with right structures, thus simplifies the downstream purification processes (Zhou et al., 2018).

Fab fragment is of smaller size than full-length antibody, and does not require post-translational modification such as glycosylation modifications, it is particularly suitable to be produced in E. coli (Walsh and Jefferis, 2006; Rezaie et al., 2017). We recently reported a method for efficient extracellular expression and purification of Fabs from E. coli, which has been optimized for many parameters (Luo et al., 2019a). Here, we provide a detailed protocol using anti-VEGF Fab as a model protein, since the anti-VEGF Fab Ranibizumab was the first approved Fab drug on the market (Danyliv et al., 2017). The process consists of three main parts: A) construction of pPhoA-Fab expression vector and transfection of host strain BL21(DE3); B) secretory soluble expression of Fab in E. coli; and C) affinity chromatography purification of Fab. We investigated the combinations with different promoters (phoA and T7) and leader peptides (STII and pelB), among them phoA-STII showed the highest yield in mass and secretion efficiency. The vector was a previously engineered pRSF (Augustine et al., 2016) plasmid containing phoA promoter, hereinafter referred to as the pPhoA plasmid. The secretory expression was induced by phosphate starvation to stimulate the function of phoA promoter (Wang et al., 2005). For affinity purification of Fab, the resin should be selected according to species and light chain types (Kappa or Lambda chain). For anti-VEGF Fab, which has human Kappa light chain, we used a prepacked Capto L column to purify it (Ulmer et al., 2019). After purification, the final product could be further analyzed for purity, yield, and bioactivities.

Generally, secretory expression in E. coli is the most suitable process for producing disulfide bond-containing antibody fragments such as Fab, Fab’, and (Fab’)2 (Ellis et al., 2017). In comparison with previous studies about extracellular expression of Fabs, our work is superior for a considerably reduced time and cost and simplified purification process, due to the use of different expression cassette designs (two separate expression cassettes, phoA promoter, STII leader sequence), host strains (BL21[DE3]) and fermentation conditions (phosphate starvation, low temperature). We have applied the protocol to prepare five Fab fragments which have been marketed successfully (anti IGF1R, anti-Her2, anti-VEGF, anti-RANKL and anti-PD-1) with different types of IgG1/IgG2 or human/humanized structures to cover as wide as possible range of Fab fragments. The results demonstrated that they were all expressed in soluble expression, and the fractions in culture medium were more than the intracellular soluble fractions or inclusion bodies content. By one-step affinity chromatography, the purity of the Fabs reached above 94%, and all products were of correct molecular weight as well as full bioactivity against their antigens. To the best of our knowledge, the current protocol is a universal technique for efficient extracellular expression, secretion and purification of Fabs in E. coli.

Materials and Reagents

Notes:

  1. All the reagents could be of other brands.
  2. Resins could be used for both AKTA equipment and manual purification, which is depend on the sample volume to handle. For example, when the sample is more than 100 ml, it saves hand-harbor using AKTA than manual purification.

  1. Inoculating loop
  2. 2 L Erlenmeyer flask
  3. 250 ml centrifuge bottles
  4. Pipettes
  5. 200 μl PCR tubes
  6. 1.5 ml Eppendorf tubes
  7. 0.22 μm filter
  8. Pipette tips
  9. E. coli strains DH5α (Shanghai Weidi Biotechnology, catalog number: DL1001)
  10. E. coli strain BL21(DE3) (Shanghai Weidi Biotechnology, catalog number: EC1002)
  11. Bgl II (concentration: 10 units/μl) (NEB, catalog number: R0144S), store at -20 °C
  12. Nde I (concentration: 20 units/μl ) (NEB, catalog number: R0111S), store at -20 °C
  13. T4 DNA Ligase (concentration: 350 units/μl) (Takara, catalog number:2011A), store at -20 °C
  14. NaCl (Sinopharm Chemical Reagent, catalog number: 10019318)
  15. Tryptone (Oxoid, catalog number: 2336957)
  16. Yeast (Oxoid, catalog number: 210408)
  17. Agar (Oxoid, catalog number: LP0011B)
  18. Kanamycin (Sinopharm Chemical Reagent, catalog number: xw253899403)
  19. PIPES (Sigma-Aldrich, catalog number: P1851-100G)
  20. (NH4)2SO4 (Sinopharm Chemical Reagent, catalog number: 10002917)
  21. MgSO4 (Sinopharm Chemical Reagent, catalog number: 20025117)
  22. D(+)-Glucose (Sinopharm Chemical Reagent, catalog number: 63005518)
  23. Tris (hydroxymethyl) aminomethane (Amresco, catalog number: 252859-100G)
  24. Methanol (Sinopharm Chemical Reagent, catalog number:1001418)
  25. Isopropanol (Sinopharm Chemical Reagent, catalog number: 80109218)
  26. Absolute alcohol (Sinopharm Chemical Reagent, catalog number: 10009218)
  27. Enhanced BCA Protein Assay Kit (Beyotime, catalog number: P0009)
  28. NaH2PO4 (Sinopharm Chemical Reagent, catalog number: 20040818) 
  29. Na2HPO4 (Sinopharm Chemical Reagent, catalog number: 20040617)
  30. K2HPO4 (Sinopharm Chemical Reagent, catalog number: 20032117)
  31. KH2PO4 (Sinopharm Chemical Reagent, catalog number: 10017618)
  32. NaOH (Sinopharm Chemical Reagent, catalog number: 10019762)
  33. Glycerol (Sinopharm Chemical Reagent, catalog number: 10010618)
  34. Citric acid (BBI, catalog number: A610055-0500) 
  35. B-PER (Thermo Fisher, catalog number: 78243)
  36. Millipore ECL (Millipore, catalog number: WBKLS0010), store at 2-8 °C
  37. Capto L resins (GE Healthcare) 
  38. KappaSelect prepacked column 1 ml (GE, catalog number: 17545811)
  39. Prestained 2x FastTaq PCR SuperMix (Miozyme, catalog number: AX111), store at -20 °C
  40. PrimeSTAR Max DNA Polymerase (Takara, catalog number: R045A), store at -20 °C
  41. AxyPrepTM PCR Cleanup Kit (Axygen, catalog number: AP-PCR-250G)
  42. AxyPrepTM Plasmid Miniprep Kit (Axygen, catalog number: AP-MN-P-250G)
  43. AxyPrepTM DNA Gel Extraction Kit (Axygen, catalog number: AP-GX-250G)
  44. PLM medium (see Recipes)
  45. Buffer A (see Recipes)
  46. Buffer B (see Recipes)
  47. TAE buffer (see Recipes)

Equipment

  1. BS 210S electronic balance (Sartorius)
  2. Electronic analytical balance (Mettler Toledo)
  3. Vertical protein electrophoresis tank (Bio-Rad)
  4. Tanon GIS Gel Imaging Analysis System (Tanon)
  5. BECKMAN COULTER Avanti J-E Centrifuge (BECKMAN COULTER)
  6. Centrifuge (Eppendorf, Model: Centrifuge 5418R)
  7. LRH-70F biochemical incubator (TENSUC)
  8. TS-200B desktop constant temperature oscillation culture shaker (TENSUC)
  9. HH-4 constant temperature water bath (Youlian)
  10. SW-CJ-IFD type ultra-clean workbench (Sujing)
  11. HYCD-205 refrigerator freezer (Haier)
  12. -80°C cryopreservation box (Haier)
  13. Benchtop pH meter FE20K (Mettler Toledo)
  14. SS325 autoclave (Tomy)
  15. TS-2 type bleaching shaker (Qilinbeier)
  16. Milli-Q Advantage A10 UltraPure Water Meter (Millipore)
  17. Electric blast drying oven (Yiheng)
  18. Thermal Cyclers for PCR (Bio-Rad)
  19. Multi-function microplate reader Infinite M200 PRO (Tecan)
  20. AKTA Avant 150 (GE)

Software

  1. Primer 5.0

Procedure

  1. Construct of anti-VEGF Fab expressing plasmids and transformation of E. coli host strain
    Notes:
    1. According to the Tm of the primers and the polymerases, set appropriate PCR reaction conditions, and select proper restriction enzymes depending on the restriction sites of the plasmids.
    2. The DNA sequence can be amplified from a template or synthesized by companies such as General Biosystems (Chuzhou, Anhui, China) we used.
    3. The backbone plasmid pPhoA can be derived from commercial plasmids such as pRSF-Duet by inserting dual phoA promoters followed by STII signal peptides.
    4. The transformation approach adapts to all the transformation mentioned in this protocol.
    1. According to the process shown in Figure 1, synthesize the entire DNA fragment encoding VL-CL, phoA promoter, STII peptide and VH-CH1 of anti-VEGF Fab expression cassette according to the published sequences. 
    2. Analyze the sequences of backbone vector and Fab fragments to determine the sites for double restriction enzymes digestion and ligation.
      Note: If there are no appreciated sites, recombinase mediated homology infusion method can be used.
    3. Design the primers with the selected restriction enzymes using Primer 5.0 software. To simplify the insertion of Fab fragments into the vector, it’s optimal to synthesize STII-VL-CL-phoA-STII-VH-CH1 by a company (for example General Biosystems, Chuzhou, Anhui, China).
      Note: To insert the target fragment into the backbone vector, we firstly determined the restriction enzymes to produce pairing sticky ends. We chose Nde I which holds the translational starting codon ATG as the upstream site. For the downstream site, it’s fine to choose any one listed in the multiple clone sites of the vector. In our study, we chose Bgl II, and the vector could be digested by Nde I and Bgl II to get the linear backbone. However, the target fragment is rather long (about 2 kb), and there are another two Bgl II sites, making it impossible to digest the target fragment with Bgl II. Therefore, we used Bsa I enzyme and designed its sticky ends the same with that of Bgl II. Then we designed the forward primer with an Nde I site and the reverse primer with a Bsa I site to amplify the fragment from the synthesized template. Finally the Nde I and Bsa I enzymes digested fragment was ligated with the Nde I and Bgl II enzymes digested backbone to construct the recombinant plasmid expressing anti-VEGF Fab.
    4. Extract backbone vector.
    5. Prepare target fragments of STII-VL-CL-phoA-STII-VH-CH1.
      1. Amplify STII-VL-CL-phoA-STII-VH-CH1 fragments with above-designed primers (Table 1) by PCR.
        1. Add the reaction reagents in 200 μl PCR tubes as shown in Table 2.

          Table 1. Sequences of primers used in PCR or sequencing

          *Primers M1 and M2 hold Nde I and Bsa I digestion sites, respectively.

          Table 2. PCR reaction recipes for amplifying STII-VL-CL-phoA-STII-VH-CH1 fragments


        2. Set the PCR program to be: denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s (according to the manual of PrimeSTAR Max, the annealing temperature could be set at 55 °C when the calculated Tm values are around 60 °C) and extension at 72 °C for 30 s (the extension time depends on the polymerase as well as the length of the target fragment, here the target fragment is about 2 kb), recycling for 35 cycles.
      2. Digest the STII-VL-CL-phoA-STII-VH-CH1 PCR products with Nde I and Bsa I, and digest the backbone vectors with double enzymes Bgl II and Nde I to get desired sticky ends.
        1. Add the reaction reagents in a 1.5 ml Eppendorf tubes as Table 3.

          Table 3. Double enzyme digestion reaction recipes


        2. Incubate the mixture in a 37 °C water bath for 1 h.
      3. Separate the digested PCR fragments or backbone vectors by a 1% DNA agarose gel electrophoresis.
      4. Retrieve the target bands from the gel with the AxyPrepTM DNA Gel Extraction Kit. 
    6. Ligate the fragments onto the backbone vector and then transform them into E. coli.
      1. Add the reaction reagents in 200 μl PCR tubes as listed in Table 4.

        Table 4. Ligation reaction recipes


      2. Incubate the mixture in the thermal cycler at 16 °C for 30 min.
      3. Transform the E. coli strain DH5α with the ligated mixture following the procedures as described below.
      4. Place the E. coli competent cells stored at -80 °C on ice to thaw.
      5. Add 10 μl of ligated products into the melted E. coli competent cells, incubate on ice for 20 min.
      6. Heat the mixture in a preheated 42 °C water bath for 80 s, followed by another 2 min incubation on ice.
      7. Add 600 μl of fresh LB broth to the tube in a clean bench.
      8. Incubate the bacteria at 37 °C, 220 rpm for 40 min in a constant temperature shaker.
      9. Centrifuge the tube at 2,150 x g for 5 min, discard most of the supernatant, retain about 200 μl of the liquid, and re-suspend the bacteria in the clean bench.
      10. Then spread it on a pre-prepared LB plate (include 100 μg/ml kanamycin).
      11. Incubate the plate upside-down in a 37 °C incubator overnight (Placing the plate upside down can prevent condensation from dripping onto the medium, avoiding contamination, and facilitating the growth, reproducibility and colony counting), then verify the clones with correct insertion.
        1. Pick a single clone in 5 μl sterilized ddH2O for colony PCR verification using the primers listed in Table 1.
        2. Add the reaction system in 200 μl PCR tubes as Table 5.

          Table 5. Colony PCR reaction recipes


        3. Set the PCR program to be: denaturation at 95 °C for 10 s, annealing at 54 °C for 5 s and extension at 72 °C for 30 s and together for 35 cycles. The primers were listed in Table 1.
        4. Use 1% DNA agarose gel electrophoresis using TAE buffer (Recipe 4) to verify the PCR products.
        5. The remaining of the clones with positive PCR fragments were further cultured in LB broth for sequencing to confirm the correct insertion of desired Fab sequences.

      12. Confirm the correct insertion of STII-VL-CL-phoA-STII- VH-CH1 fragments by sequencing, and get the recombinant pPhoA-Fab plasmid as shown in Figure 1.


        Figure 1. Construction process of pPhoA-Fab plasmid. STII-VL-CL-phoA-STII- VH-CH1 fragment was synthesized, PCR amplified, digested by Nde I and Bsa I, and ligated with backbone vector digested by Nde I and Bgl II, to obtain the recombinant plasmid pPhoA-Fab.

      13. Culture the DH5α clone that sequenced to be with the correct insert overnight to extract recombinant plasmid pPhoA-Fab as shown in Figure 1 by the AxyPrepTM Plasmid Miniprep Kit.
      14. Using the constructed pPhoA-Fab to further transform BL21 (DE3) using the same heat shock procedures (Steps A6e to A6l), and the successfully transformed clone is used as the expression host strain for anti-VEGF Fab expression.
      15. Make the transformed bacteria DH5α and BL21 (DE3) aliquots, add glycerol to a final concentration of 20% and store at -80°C for future use.

  2. Extracellular expression of anti-VEGF Fab
    1. Take a vial of BL21 (DE3) containing pPhoA-Fab from -80 °C, put it on ice for 5 min.
    2. Transfer the thawed BL21 (DE3) to an LB agar plate (containing 100 μg/ml kanamycin) using inoculating loop, incubate at 37 °C overnight.
    3. Pick a single clone from the LB plate, and inoculate it into a tube containing 5 ml of LB broth (containing 100 μg/ml kanamycin).
      Notes:
      1. It takes about 12-14 h for preculture, so it’s best to carry out this step in the evening to ensure using fresh E.coli to inoculate. 
      2. The ingredients of LB media of various brands are consistent, so any qualified brand of LB medium can be used.
    4. Culture the bacteria in the tube in a shaking incubator at 37 °C, 220 rpm for about 12-14 h. No more than 16 h.
    5. Inoculate 5 ml culture into 800 ml of fresh LB broth containing 100 μg/ml kanamycin.
      Note: Use a 2 L Erlenmeyer flask. The LB medium volume should not occupy over 40% of the full capacity of the container.
    6. Culture the bacteria at 37 °C, 220 rpm until OD600 reaches 0.8-1.0.
    7. Transfer the bacteria to a sterilized 250 ml centrifuge bottles to pellet the bacteria at room temperature by centrifugation at 4,000 x g for 10 min.
      Note: This step should be performed in the clean bench.
    8. Discard the supernatant, re-suspend the bacterial pellet with 800 ml sterilized PLM medium.
      Note: PLM medium is chemically modified and contains no phosphate. This step should be performed in the clean bench. Here take 1 ml of culture for analysis, marked as ‘Pre’. 
    9. Incubate the bacteria in PLM medium overnight at a temperature of 20 °C with a shaking speed of 200 rpm for about 12-14 h. The absence of phosphate in PLM medium promotes protein induction.
    10. After induction, quickly raise the incubation temperature to 59 °C in the water bath and incubate for 1 h to improve Fab yields.
      Note: This step can improve the extracellular yield for some level, it is also OK to skip it.
    11. Then, quickly cool down the culture to room temperature in a water bath and centrifuge at 3,500 x g for 10 min to separate media and bacterial pellet, the expressed anti-VEGF Fab should mostly remain in the medium. 
    12. Collect the culture medium and further centrifuge at 12,000 x g for 10 min to further remove the contaminants.
      Note: Immediately before centrifugation, take 2 aliquots of culture for analysis, 1 ml for each, marked as ‘Ind’.

  3. Purification of anti-VEGF Fab
    Notes:
    1. The Capto L binds to kappa light chains of type I, III, and IV of human IgG but not type II. 
    2. KappaSelect resin can be used to purify protein with a type II IgG kappa light chain. The purification process is similar to that of Capto L, but using PBS buffer of pH 7.4 as Buffer A, and 0.1 M glycine buffer of pH 2.7 as Buffer B.
    1. Filter the expression medium through a 0.22 μm filter for subsequent column chromatography purification steps.
    2. Prepare AKTA Avant 150 by washing the A, B pumps and tubes with 20% ethanol at 2 ml/min until the UV280 reaches baseline. 
    3. Assemble the prepacked Capto L column with a column volume (CV) of 1 ml at a flow rate of 0.5 ml/min, and continue to wash the system with 20% ethanol at 2 ml/min until the UV 280 nm and Cond baselines are stable.
    4. Then, repeat the washing with ddH2O with the same procedure until the UV 280 nm and Cond baselines are stable.
    5. Place the B1 tube head in Buffer B and fill the tube with Buffer B.
    6. Place the A1 tube head in Buffer A, and equilibrate the system and column for 10-20 CV until the UV 280 nm and Cond reach baseline.
    7. Load the sample onto the column at a flow rate of 0.5 ml/min while collecting flow through.
      Note: Collect 1 ml of flow through for analysis, marked as ‘FL’.
    8. After loading, wash the column with Buffer A until the UV 280 nm reaches the baseline. 
    9. Set the B pump solution ratio to 100%, elute the target protein by Buffer B with a flow rate of 1.0 ml/min, and collect the target protein when there are UV280 peaks.
    10. Immediately adjust the pH of the collected sample with 1 M Tris buffer (pH 9.0) to prevent protein inactivation.
      Note: Take eluted protein samples for analysis, marked as ‘E’.
    11. After the baseline of the elution is stable, flush the tubing with 10 to 20 CV ddH2O until the baselines of UV and Cond are stable. 
    12. Then, rinse the tubing and column with 15 mM NaOH to clean the column. After the UV280 baseline is stabilized, wash the system, column, and tubes with ddH2O and 20% ethanol successively. 
    13. Finally, fill the pipeline with 20% ethanol and disassemble the pre-packed column, which should also be kept in 20% ethanol. 
    14. Test the collected protein by SDS-PAGE and Western blot (WB).


      Figure 2. Scheme of the expression and purification processes for Fab production (Luo et al., 2019a)  

Data analysis

  1. Take one aliquot of the collected sample ‘Ind’ and centrifuge at 3,500 x g for 10 min to separate medium (marked as ‘Medi’), and use B-PER to incubate the pellet for 15 min at room temperature to release the intracellular soluble proteins.
  2. Centrifuge at 12,000 x g for 30 min at 4 °C to separate intracellular soluble (marked as ‘Sup’) and insoluble (marked as ‘IB’) fractions. 
  3. To investigate the distribution of anti-VEGF Fab expression, analyze all samples Pre, Ind, Medi, Sup, and IB by SDS-PAGE (Figure 3A) and WB (Figure 3B). Use a goat anti-human kappa light chains antibody as the capture antibody, and rabbit anti-goat IgG antibody conjugated with HRP as the second antibody. 
  4. The purification process (Figure 4A) can be analyzed by SDS-PAGE of samples Medi, FL, and E (Figure 4B).
  5. For the purity estimation of the Fab antibody, the product can be determined by size exclusion high performance liquid chromatography (SEC-HPLC) using TSK G2000SWXL column (5 μm, 0.78 x 300 mm) at an Agilent 1260 HPLC system. The purity of each Fab can be calculated as the area percentage of the corresponding peak detected.
  6. The molecular weight of the Fab can be accurately measured using an ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometer (MALDI-TOF MS). 
  7. The affinity between the Fab and the specific antigen can be tested using the ForteBio Octet RED96e system.


    Figure 3. Expression of anti-VEGF Fab in E. coli. A. SDS-PAGE; B. Western blot. Most of anti-VEGF Fab was secreted into the culture medium. Lane Pre: bacteria before induction; Ind: bacteria after induction; Medi: Medium; Sup: intracellular soluble fraction; IB: Inclusion bodies. The samples charged in the same volume and all samples were non-reducing. The star (★) indicates the predicted size of Fab at about 40 kDa (Luo et al., 2019b).

    The samples charged in the same volume and all samples were non-reducing. The star (★) indicates the predicted size of Fab at about 40 kDa (Luo et al., 2019b).


    Figure 4. Purification of anti-VEGF Fab by Capto L affinity chromatography. A. Eluted chromatography spectrum. Red curve: UV absorbance at 280 nm; Blue curve: Conductivity. B. SDS-PAGE. Lane Medi: Pre-loading sample (medium); FL: flow through; E(NR): non-reducing elutant; E(R): reduced elutant. The star (★) indicates the non-reducing Fab; the diamond (◆) indicates the educed fragments of LC(▲) and HC(▲) (Luo et al., 2019b).

Recipes

  1. PLM medium
    7.5 mM (NH4)2SO4
    0.4 mM MgSO4
    11 mM glucose
    111 mM PIPES
    Adjust to pH 7.0
    Note: PLM medium should be steam-sterilized or filter-sterilized immediately after preparation.
  2. Buffer A
    50 mM citric acid
    50 mM sodium citrate
    Adjust to pH 6.5
    Note: The buffer should be filtered through a 0.22 μm filter and used within one week. 
  3. Buffer B
    50 mM citric acid
    50 mM sodium citrate
    Adjust to pH 2.3
    Note: The buffer should be filtered through a 0.22 μm filter and used within one week. 
  4. TAE buffer
    40 mM Tris-acetate, pH 8.5
    1 mM EDTA

Acknowledgments

This work was in collaboration with Jecho Labs, and a US patent was applied jointly (CAGLIERO, Cedric; BURNETTE, Andrew; XIE, Yueqing; JIANG, Hua; ZHU, Jianwei; LU, Huili; LUO, Manyu. Method for Preparing Recombinant Protein from Bacterium and Composition Containing the Same. United States Patent Application, Date: Dec. 31, 2018, Application No. 16/237265). The work was also supported in part by the National Natural Science Foundation of China (No. 81773621 to Zhu J.), the Science and Technology Commission of Shanghai Municipality (No. 17431904500 & 17ZR1413700 to Lu H.). We thank all the authors of our original research article (Luo et al., 2019a Appl Microbiol Biotechnol).

Competing interests

The authors declare no conflicts of interest with the contents of this article.

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  15. Zhou, Y., Lu, Z., Wang, X., Selvaraj, J. N. and Zhang, G. (2018). Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 102(4): 1545-1556.

简介

与全长igg相比,抗原结合片段(fab)较小,不需要复杂的翻译后修饰。因此,用E.E.E.E.大肠杆菌(大肠杆菌)表达体系可以有效地生产Fab。然而,含有外源蛋白的二硫键(包括fab)往往在大肠杆菌中形成不溶性包涵体,成为该系统表达外源蛋白的瓶颈。蛋白质在胞外或周质中的分泌表达是防止包涵体形成、提高大肠杆菌生产fabs效率的有效途径。细胞外表达特别有趣,因为它将产物释放到培养基中,而周质表达的产量仅限于周质空间。此外,细胞外表达可以直接从培养上清液中提取蛋白质,省去细胞裂解过程,减少宿主细胞蛋白质或dna的污染。以抗vegf-fab为例,我们提供了一个基于碱性磷酸酶(phoa)启动子和热稳定肠毒素ii(stii)先导序列的方案。利用磷酸饥饿诱导fabs的分泌表达,该方法可普遍用于fabs的高效生产。
【背景】大肠杆菌具有遗传背景清晰、操作简便、生产成本低廉等优点,被广泛应用于外源基因的表达,尤其是分子量低、构象结构简单的外源基因的表达(Gupta和Shukla,2017)。外源蛋白在大肠杆菌中的表达主要分为包涵体表达、胞内可溶性表达和胞外分泌三类(Jalaliard,2013;Gupta and Shukla,2017;Zhou等人,2018)。目前,哺乳动物来源的外源蛋白大多以包涵体或可溶性的形式在大肠杆菌胞内表达。但这两种形式对于后续过程都有各自的缺点。包涵体需要经过繁琐的变性和复性以恢复具有正确复性结构的目标蛋白,并且这些过程往往导致生物活性和产量的降低(Panda等人,2003;Nelson和Reichert,2009)。E.E.E.大肠杆菌的周质空间可为二硫键形成提供有利的氧化环境,但产量通常受周质空间的容量和信号肽的超前能力的限制(LoSPTAN等)2012;埃利斯2017年)。与上述两个过程相比,胞外蛋白表达不受胞内空间的限制,可以方便地富集结构正确的靶蛋白,从而简化了下游纯化过程(Zhou等人,2018)。



fab片段比全长抗体小,不需要糖基化等翻译后修饰,特别适合在大肠杆菌中生产(walsh and jefferis,2006;rezaieet al.,2017)。我们最近报道了一种高效表达和纯化大肠杆菌fabs的方法,该方法已经优化了许多参数(luo等人,2009年a)。在这里,我们提供了一个详细的协议,使用抗VEGF-Fab作为模型蛋白,因为抗VEGF Fab雷尼珠单抗是市场上第一个批准的Fab药物(DANILIV等。,2017)。该过程主要包括三个部分:a)pphoa-fab表达载体的构建和宿主菌bl21(de3)的转染;b)fab在大肠杆菌中的分泌可溶性表达;c)fab的亲和层析纯化。我们研究了不同启动子(phoa和t7)和先导肽(stii和pelb)的组合,其中phoa stii的质量和分泌效率最高。该载体是以前构建的PRSF(奥古斯丁等.,2016)含有PHA启动子的质粒,以下简称PPHOA质粒。磷酸饥饿诱导分泌表达促进PHA启动子的功能(王等. 2005)。对于fab的亲和纯化,应根据种类和轻链类型(kappa或lambda链)选择树脂。对于具有人kappa轻链的抗vegf fab,我们使用预先包装的capto l柱纯化(ulmer等,2019)。纯化后的产物可进一步进行纯度、收率和生物活性分析。



一般来说,大肠杆菌中的分泌表达是产生含有二硫键的抗体片段(如Fab、Fab'和(Fab')2)的最合适的方法(Ellis等人,2017年)。与以往有关fabs细胞外表达的研究相比,由于采用了不同的表达盒设计(两个单独的表达盒,phoa启动子,stii先导序列),宿主菌株(bl21[de3])和发酵条件(磷酸盐饥饿,低温)。我们应用该方案制备了5个已成功上市的fab片段(抗igf1r、抗her2、抗vegf、抗rankl和抗pd-1),它们具有不同类型的igg1/igg2或人源化结构,以覆盖尽可能广泛的fab片段。结果表明,它们均以可溶性表达,且培养基中可溶性组分含量大于细胞内可溶性组分或包涵体含量。经一步亲和层析,产物纯度达到94%以上,分子量正确,对抗原具有完全的生物活性。据我们所知,目前的方案是一种在大肠杆菌中高效表达、分泌和纯化fabs的通用技术。

关键字:Fab, PhoA, STII, 细胞外合成, 分泌

材料和试剂

注意:

  1. 所有试剂都可以是其他品牌的。
  2. 树脂既可用于AKTA设备,也可用于人工净化,这取决于处理样品的体积。例如,当样品超过100毫升时,使用AKTA比手动净化节省了手动港。
< >
  1. 接种回路
  2. 2 L锥形烧瓶
  3. 250毫升离心瓶
  4. 微量加样器
  5. 200μl PCR管
  6. 1.5毫升Eppendorf试管
  7. 0.22μm过滤器
  8. 吸管尖头
  9. 大肠杆菌菌株DH5α(上海维迪生物技术,目录号:DL1001)
  10. 大肠杆菌菌株BL21(DE3)(上海维迪生物技术,目录号:EC1002)
  11. bglii(浓度:10单位/μl)(neb,目录号:r0144s),储存于-20°C
  12. 无损检测i(浓度:20单位/μl)(NEB,目录号:R0111S),储存于-20°C
  13. T4 DNA连接酶(浓度:350单位/μL)(Takara,目录号:2011a),储存于-20°C
  14. 氯化钠(国药化学试剂,目录号:10019318)
  15. 胰蛋白胨(类氧化合物,目录号:2336957)
  16. 酵母(Oxoid,目录号:210408)
  17. 琼脂(oxoid,目录号:lp0011b)
  18. 卡那霉素(国药化学试剂,目录号:XW253899403)
  19. 管道(Sigma-Aldrich,目录号:p1851-100g)
  20. (NH4)2SO4(国药化学试剂,目录号:10002917)
  21. mgso4(国药化学试剂,目录号:20025117)
  22. D(+)-葡萄糖(国药化学试剂,目录号:63005518)
  23. 三羟甲基氨基甲烷(Amresco,目录号:252859-100g)
  24. 甲醇(国药化学试剂,目录号:1001418)
  25. 异丙醇(国药化学试剂,目录号:80109218)
  26. 无水乙醇(国药化学试剂,目录号:10009218)
  27. 增强型bca蛋白检测试剂盒(beyotime,目录号:p0009)
  28. nah2po4(国药化学试剂,目录号:20040818)
  29. Na2HPO4(国药化学试剂,目录号:20040617)
  30. K2HPO4(国药化学试剂,目录号:20032117)
  31. kh2po4(国药化学试剂,目录号:10017618)
  32. NaOH(国药化学试剂,目录号:10019762)
  33. 甘油(国药化学试剂,目录号:10010618)
  34. 柠檬酸(BBI,目录号:A610055-0500)
  35. B-PER(赛默飞世尔,目录号:78243)
  36. Millipore ECL(Millipore,目录号:WBKLS0010),2-8°C保存
  37. Capto L树脂(GE Healthcare)
  38. Kappaselect预包装柱1 ml(GE,目录号:17545811)
  39. 预处理2x FastTaq PCR Supermix(Miozyme,目录号:AX111),储存于-20°C
  40. Primestar Max DNA聚合酶(Takara,目录号:R045A),储存于-20°C
  41. AXYPREPTMPCR清洗试剂盒(AXYGEN,目录号:AP-PCR-250G)
  42. axypreptm质粒微制备试剂盒(axygen,目录号:ap-mn-p-250g)
  43. AXYPREP TM DNA凝胶提取试剂盒(AXYGEN,目录号:AP-GX-250G)
  44. PLM培养基(见食谱)
  45. 缓冲区A(见配方)
  46. 缓冲区B(见配方)
  47. TAE缓冲液(见配方)

设备

  1. BS 210S电子天平(Sartorius)
  2. 电子分析天平(梅特勒-托莱多)
  3. 垂直蛋白质电泳槽(Bio-Rad)
  4. GIS凝胶成像分析系统(Ⅱ)
  5. 贝克曼库尔特-阿凡提J-E离心机(贝克曼库尔特)
  6. 离心机(Eppendorf,型号:离心机5418R)
  7. lrh-70f生化培养箱(tensuc)
  8. ts-200b台式恒温振荡培养瓶(tensuc)
  9. HH-4恒温水浴(友联)
  10. sw-cj-ifd型超净工作台(苏静)
  11. 海尔hycd-205冰箱
  12. -80°C冷冻箱(海尔)
  13. 台式pH计FE20K(梅特勒-托莱多)
  14. SS325高压灭菌器(tomy)
  15. TS-2型漂白摇床(齐林贝尔)
  16. Milli-Q Advantage A10超纯水表(微孔)
  17. 电鼓风干燥炉(一恒)
  18. PCR用热循环器(Bio-Rad)
  19. 多功能微型读板器Infinite M200 Pro(帝肯)
  20. Akta Avant 150(通用电气)

软件

  1. 引物5

程序

  1. 抗vegf-fab表达质粒的构建及其对大肠杆菌的转化 注意:
    1. 根据引物和聚合酶的TM,设置合适的PCR反应条件,根据质粒的限制性位点选择合适的限制性内切酶。
    2. E.> DNA序列可以从模板中扩增出来,也可以由通用生物系统公司(滁州、安徽、中国)等公司合成。
    3. <> EM质粒pPHOA可通过插入双PoA启动子和STII信号肽而从PRSF二元等商业质粒中获得。
    4. 转换方法适用于本协议中提到的所有转换。
    1. 根据图1所示的过程,根据公布的序列合成编码抗vegf fab表达盒vl-cl、phoa启动子、stii肽和vh-ch1的整个dna片段。
    2. 分析骨干载体和fab片段序列,确定双酶切和连接位点。< > E.>注释:如果不存在受欢迎位点,则可以使用重组酶介导的同源灌注方法。
    3. 用primer 5.0软件设计具有所选限制性内切酶的引物。为了简化fab片段插入载体的过程,最好由一家公司(例如中国安徽滁州通用生物系统公司)合成stii-vl-cl-phoa-stii-vh-ch1。
      注:为了将目标片段插入到骨干载体中,我们首先确定了产生成对粘端的限制性内切酶。我们选择了ndei,它将翻译起始密码子atg作为上游位点。对于下游站点,可以选择列在向量的多个克隆站点中的任何一个。在我们的研究中,我们选择了bgli i,载体可以被ndei和 bglii得到线性主干。然而,靶片段相当长(约2kb),并且还有另外两个bglii位点,使得用bglii无法消化靶片段。因此,我们使用bsaⅠ酶,设计了与bgl相同的粘端。然后我们设计了具有ndei位点的正向引物和具有bsa i位点的反向引物,以扩增合成模板的片段。最后,< < NE> < Bsa >酶消化片段与<< E/E> E> < NDE I和 Bgl EM> II酶消化主干,构建表达VEGF-Fab的重组质粒。
    4. 提取主干向量。
    5. 制备stii-vl-cl-phoa-stii-vh-ch1的靶片段。
      1. 用上述设计的引物(表1)扩增stii-vl-cl-phoa-stii-vh-ch1片段。
        1. 将反应试剂加入200μl PCR管中,如表2所示。
          < > 表1。用于PCR或测序的引物序列
          *引物m1和m2分别具有ndei和bsai消化位点。
          < > 表2。扩增stii-vl-cl-phoa-stii-vh-ch1片段的pcr反应配方
          < >
        2. 将PCR程序设置为:98℃变性10s,55℃退火5s(根据Primestar Max手册,当计算得到的tm值在60℃左右时,退火温度可以设定在55℃,72℃延长30s(延长时间取决于聚合酶和靶片段的长度,这里靶片段约为2kb),循环35个周期。
      2. 用ndei和bsai消化stii-vl-cl-phoa-stii-vh-ch1 pcr产物,用双酶bglii和ndei消化骨干载体,得到所需的粘端。
        1. 将反应试剂加入1.5mL的Eppendorf试管中,如表3所示。
          < > 表3。双酶消化反应配方
          < >
        2. 将混合物在37°C水浴中培养1h。
      3. 用1%琼脂糖凝胶电泳分离消化的pcr片段或主链。
      4. 用AXYPREP TM DNA凝胶提取试剂盒从凝胶中回收靶条带。
    6. 将片段连接到主干载体上,然后将其转化为大肠杆菌。
      1. 如表4所示,将反应试剂加入200μl PCR管中。
        < > 表4。结扎反应配方
        < >
      2. 将混合物在16°C的热循环器中培养30分钟。
      3. 按照以下步骤用结扎混合物转化大肠杆菌dh5α株。
      4. 将大肠杆菌活性细胞放在-80°C的冰上解冻。
      5. 将10μl结扎产物加入溶化的大肠杆菌活性细胞中,在冰上孵育20分钟。
      6. 在预热的42°C水浴中加热混合物80秒,然后在冰上再培养2分钟。
      7. 在干净的工作台上向试管中加入600μl新鲜lb肉汤。
      8. 将细菌在37°C、220转/分的恒温摇瓶中培养40分钟。
      9. 以2150x g离心管5分钟,丢弃大部分上清液,保留约200μl液体,并将细菌重新悬浮在干净的工作台上。
      10. 然后将其涂在预先准备好的lb板上(包括100μg/ml卡那霉素)。
      11. 在37°C培养箱中将板倒置过夜(放置倒置的板可以防止凝结物滴到培养基上,避免污染,促进生长、重复性和菌落计数),然后验证克隆的正确插入。
        1. 用表1所列的引物,在5μl灭菌ddh2o中挑选一个克隆进行菌落pcr验证。
        2. 将反应体系加入200μl PCR管中,如表5所示。
          < > 表5。菌落PCR反应配方
          < >
        3. 将PCR程序设置为:95℃变性10s,54℃退火5s,72℃延长30s,共35个周期。引物列于表1。
        4. 采用1% DNA琼脂糖凝胶电泳(TAE缓冲液)(配方4)对PCR产物进行验证。
        5. 其余pcr阳性克隆进一步在lb培养基中培养,以确定所需fab序列的正确插入。< >
          < >
      12. 通过测序确认stii vl cl phoa stii-vh-ch1片段的正确插入,得到重组pphoa fab质粒,如图1所示。< > < >
        图1。pOPHA Fab质粒的构建过程,合成了 NDE I和 Bgl II的骨架载体连接,获得重组质粒pOPHA Fab。< >
      13. 用axypreptm质粒miniprep试剂盒培养测序正确插入的dh5α克隆,以提取重组质粒pphoa fab,如图1所示。
      14. 利用构建的pphoa-fab,采用相同的热休克程序(步骤a6e至a6l)进一步转化bl21(de3),成功转化的克隆作为抗vegf-fab表达的表达宿主菌株。
      15. 将转化菌dh5α和bl21(de3)等分,加入甘油至最终浓度20%,并在-80℃下保存以备将来使用。
        < >
  2. 抗vegf-fab的细胞外表达
    1. 从-80°C取一小瓶含有pphoa fab的bl21(de3),放在冰上5分钟。
    2. 用接种环将解冻的bl21(de3)转移到lb琼脂板(含100μg/ml卡那霉素)中,在37℃下培养过夜。
    3. 从lb板上取一个克隆,接种到含有5ml lb肉汤(含100μg/ml卡那霉素)的试管中。< > 注:
      1. 预培养大约需要12-14小时,因此最好在晚上执行此步骤,以确保使用新鲜大肠杆菌进行接种。
      2. 不同品牌的LB培养基成分是一致的,因此可以使用任何合格的LB培养基。
    4. 细菌在37°C,220转/分的振荡培养箱中培养12-14小时,不超过16小时。
    5. 将5ml培养液接种于含卡那霉素100μg/ml的新鲜lb肉汤800ml中。< > 注意:使用2L锥形烧瓶。磅中等体积不应占容器总容量的40%以上。
    6. 在37°C,220转/分的温度下培养细菌,直到od600达到0.8-1.0。
    7. 将细菌转移到一个已消毒的250毫升离心瓶中,在室温下以4000x g离心10分钟使细菌颗粒化。
      注意:此步骤应在干净的工作台上执行。
    8. 弃培养上清液,用800 ml灭菌PLM培养基再悬浮细菌颗粒。< > 注:PLM培养基经化学改性,不含磷酸盐。此步骤应在干净的工作台上执行。这里取1毫升培养基进行分析,标记为“pre”。
    9. 在PLM培养基中培养细菌,温度为20°C,振荡速度为200转/分钟,约12-14h。PLM培养基中磷酸的缺乏促进了蛋白质的诱导。
    10. 诱导后,在水浴中迅速将孵育温度提高到59℃,孵育1h以提高fab产率。< > 注意:这一步可以在一定程度上提高细胞外产量,跳过也可以。
    11. 然后,在水浴中迅速将培养物冷却至室温,并在3500x g下离心10分钟,分离培养基和细菌颗粒,表达的抗vegf fab大部分应留在培养基中。
    12. 收集培养基并在12000 x g下进一步离心10分钟,以进一步去除污染物。
      注意:离心前,取2份培养液进行分析,每份1毫升,标记为“ind”。
      < >
  3. 抗vegf fab的纯化 注:
    1. Capto L与人IgG的I、III和IV型Kappa轻链结合,但不与II型结合。
    2. kappaselect树脂可用于纯化具有ii型igg-kappa轻链的蛋白质。纯化过程与capto l相似,但以ph 7.4的pbs缓冲液为缓冲液a,ph 2.7的0.1m甘氨酸缓冲液为缓冲液b。
    1. 通过0.22μm过滤器过滤表达培养基,以进行随后的柱层析纯化步骤。
    2. 用20%乙醇以2毫升/分钟的速度清洗A、B泵和试管,直到UV280达到基线,制备AKTA Avant 150。
    3. 以0.5ml/min的流速将预先包装好的Capto L柱组装成1ml的柱体积(Cv),并继续以2ml/min的速度用20%乙醇清洗系统,直到紫外线280 nm和条件基线稳定。
    4. 然后,用相同的程序用ddh2o重复洗涤,直到uv 280 nm和cond基线稳定。
    5. 将B1管头放入缓冲器B中,并用缓冲器B填充管。
    6. 将a1管头置于缓冲液a中,并使系统和柱平衡10-20 cv,直到uv 280 nm和cond达到基线。
    7. 收集流经的样品时,以0.5毫升/分钟的流速将样品装载到柱上。< > 注意:收集1毫升流动液进行分析,标记为“FL”。
    8. 加载后,用缓冲液A清洗色谱柱,直到紫外280 nm达到基线。
    9. 设b泵液比为100%,用缓冲液b以1.0ml/min的流速洗脱目标蛋白,当有uv280峰时收集目标蛋白。
    10. 立即用1 M Tris缓冲液(pH 9.0)调节采集样品的pH值,以防止蛋白质失活。< > 注:取洗脱蛋白样品进行分析,标记为“E”。
    11. 在洗脱基线稳定后,用10至20 cv ddh2o冲洗管子,直到紫外线和cond基线稳定。
    12. 然后,用15 mm NaOH冲洗管和柱,以清洁柱。在uv280基线稳定后,依次用ddh2o和20%乙醇清洗系统、色谱柱和试管。
    13. 最后,用20%乙醇填充管道并拆卸预包装的塔,该塔也应保存在20%乙醇中。
    14. 用SDS-PAGE和western blot(WB)对收集的蛋白质进行检测。
      < >
      图2。fab生产的表达和纯化工艺方案(Luo等,2019a)

数据分析

  1. 从采集的样品“ind”中取一份,在3500x g下离心10分钟,分离培养基(标记为“medi”),并使用b-per在室温下培养15分钟,以释放细胞内可溶性蛋白质。
  2. 在12000x g下离心30分钟,在4°C下分离细胞内可溶性(标记为“sup”)和不可溶性(标记为“ib”)部分。
  3. 为了研究抗血管内皮生长因子Fab表达的分布,通过SDS-PAGE(图3A)和WB(图3B)分析所有样本Pre、Ind、Medi、Sup和IB。以羊抗人kappa轻链抗体作为捕获抗体,兔抗羊igg抗体与hrp结合作为第二抗体。
  4. 纯化过程(图4a)可以通过SDS-PAGE分析,FL,E和E(图4B)。
  5. 为确定Fab抗体的纯度,可在AgLunter 1260 HPLC系统中用TSK G2000 SWXL柱(5μm,0.78×300 mm)进行大小排阻高效液相色谱(SEC-HPLC)测定。每个Fab的纯度可以计算为检测到对应峰的面积百分比。
  6. 用超高效液相色谱-四极杆飞行时间质谱仪(MALDI-TOF MS)可以精确地测量FAB的分子量。
  7. Fab和特定抗原之间的亲和性可以使用FurtBioOcTeTeRe96e系统进行测试。< > < >
    图3。抗vegf fab在大肠杆菌中的表达。a.sds-page;b.western blot。大部分抗VEGF-Fab分泌到培养基中。lane pre:诱导前细菌;ind:诱导后细菌;medi:培养基;sup:细胞内可溶性部分;ib:包涵体。< > < > 在相同体积中充电,所有样品均为非还原性。星号(★)表示fab在40kda左右的预测大小(luo等人,2019b)。 < >
    图4。capto l亲和层析纯化抗vegf-fab。a.洗脱色谱光谱。红色曲线:280nm处的紫外吸收;蓝色曲线:电导率。B.SDS-PAGE。Lane Medi:预载样品(介质);FL:流经;E(NR):非还原洗脱剂;E(R):还原洗脱剂。星号(★)表示非还原fab,菱形(◆)表示lc(??)和hc(??)的导出碎片(luo等,2009年b)。

食谱

  1. PLM培养基
    7.5毫米(nh4)2so4
    0.4毫米mgso4
    11毫米葡萄糖
    111 mm管道
    调节至pH 7.0
    注:PLM培养基应在灭菌后立即灭菌或过滤灭菌。
  2. 缓冲区a
    50毫米柠檬酸
    50毫米柠檬酸钠
    调节至pH 6.5
    注意:缓冲液应通过0.22μm过滤器过滤,并在一周内使用。
  3. 缓冲区b
    50毫米柠檬酸
    50毫米柠檬酸钠
    调节至pH 2.3
    注意:缓冲液应通过0.22μm过滤器过滤,并在一周内使用。
  4. tae缓冲区
    40毫米醋酸三钠,pH值8.5
    1毫米EDTA

致谢

这项工作是与杰乔实验室合作,并共同申请了美国专利(卡格里罗、塞德里克、伯内特、安得烈、谢、乐清、JIANG、华、朱、Jianwei、鲁、Huili、罗、Manyu)。从细菌和含有重组蛋白的成分中制备重组蛋白的方法。美国专利申请,日期:2018年12月31日,申请号:16/237265)。这项工作也得到了国家自然科学基金(Zhu J. 81773621号)、上海市科学技术委员会(17431904500号和17ZR1413700对Lu H.)的支持。我们感谢我们原始研究文章的所有作者(Luo等人,2019aAppl Microbiol Biotechnol)。

相互竞争的利益

作者声明与本文内容没有利益冲突。

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引用:Wang, Z., Gao, Y., Luo, M., Cagliero, C., Jiang, H., Xie, Y., Zhu, J. and Lu, H. (2019). A PhoA-STII Based Method for Efficient Extracellular Secretion and Purification of Fab from Escherichia coli. Bio-protocol 9(18): e3370. DOI: 10.21769/BioProtoc.3370.
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