Method for preparing intermediate peptide of GLP-1 peptide or analogue thereof

Abstract

A method for preparing an intermediate peptide of GLP-1 peptide or an analogue thereof by means of an inclusion body in Escherichia coli. Provided is a tandem protein having general formula of T-G-(L-G)n.

Description

A method for preparing intermediate peptides of GLP-1 peptide or analogues Technical Field

[0001] This invention belongs to the field of peptide preparation, specifically relating to a method for preparing intermediate peptides of GLP-1 peptide or its analogues in Escherichia coli via inclusion bodies. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) is an intestinal hormone secreted by L cells, endocrine cells of the intestinal mucosa. It is secreted after eating and regulates insulin secretion in a glucose-dependent manner, inhibiting glucagon release and lowering blood glucose levels. However, the half-life of GLP-1 secreted by the human body is very short, only 1-2 minutes. After being secreted into the bloodstream, it is easily degraded by dipeptidyl peptidase-4 (DPP-4) and loses its insulin-secreting activity. To prolong the half-life of GLP-1, researchers have modified its structure, developing a series of GLP-1 analogs, GLP-1RAs (GLP-1 receptor agonists), such as smegglutide, liraglutide, abiglutide, and dulaglutide.

[0003] Smegglutide is currently produced mainly through the following three methods: 1. Chemical solid-phase synthesis, which has the disadvantages of being complex, having low yield, and containing many impurities; 2. Biological expression of 29 amino acids other than His-Aib (Arg34GLP-1(9-37)), followed by chemical linking of the dipeptide His-Aib, and then side chain linking; and 3. Biological expression of 27 amino acids other than His-Aib-Glu-Gly (Arg34GLP-1(7-37)), followed by chemical linking of the tetrapeptide His-Aib-Glu-Gly, and then side chain linking.

[0004] The biological expression of intermediates Arg34GLP-1(9-37) and Arg34GLP-1(7-37) currently mainly involves the following three technical routes: 1. Extracellular secretion expression in yeast, which has the disadvantage of long fermentation cycle and low purity of target product due to cleavage by host cell proteases; 2. Intracellular soluble expression in Escherichia coli, which has the disadvantage of low yield of target product; 3. Intracellular inclusion body expression in Escherichia coli, which has the disadvantage of low yield of target product and high production cost due to large amount of cleavage enzyme.

[0005] There is still a need in this field for bio-preparation methods of GLP-1 peptide intermediates that are high in purity, high in yield, and low in cost. Summary of the Invention

[0006] On one hand, the present invention provides a precursor peptide with the general formula: Met–precursor peptide core sequence–LysArg or ArgArg or AspAspAspAspLys (SEQ ID NO: 144), wherein the precursor peptide core sequence is selected from T1-T19 (as shown in SEQ ID NO: 112-130 respectively). In one embodiment, the precursor peptide core sequence is selected from T3, T5, T6, T8, T9, T10, T11, T12, T13, T14, T15, T16, T17, T18, and T19. In another embodiment, the precursor peptide core sequence is selected from T3, T5, T6, T8, T9, T12, T13, T14, T15, T16, and T19. In yet another embodiment, the precursor peptide core sequence is selected from T3, T9, T12, T14, T16, and T19.

[0007] In embodiments where the N-terminus is LysArg or ArgArg, the core sequence of the leader peptide is selected from T1-T4. In one embodiment, the core sequence of the leader peptide is T3.

[0008] In an embodiment where the N-terminus is AspAspAspAspLys, the core sequence of the precursor peptide is selected from T5-19. In one embodiment, the core sequence of the precursor peptide is selected from T5, T6, T8, T9, T10, T11, T12, T13, T14, T15, T16, T17, T18, and T19. In another embodiment, the core sequence of the precursor peptide is selected from T9, T12, T14, T16, and T19.

[0009] On one hand, the present invention provides a linker peptide with the general formula: LysArg or ArgArg – linker peptide core sequence – AspAspAspAspLys, wherein the linker peptide core sequence is selected from L1-L9 (as shown in SEQ ID NO: 131-135, none, and SEQ ID NO: 136-138, respectively). In one embodiment, the linker peptide core sequence is selected from L1, L2, L3, L5, L7, and L8. In another embodiment, the linker peptide core sequence is selected from L1, L2, and L8.

[0010] On one hand, the present invention provides a polypeptide construct with the general formula: T–G–(L–G) n ,in,

[0011] T is the precursor peptide of this invention.

[0012] L is the linker peptide of this invention.

[0013] G is a GLP-1 peptide or its analogue or intermediate peptide.

[0014] n is a natural number.

[0015] In one embodiment, the GLP-1 peptide or its analogue or its intermediate peptide is G5 (as shown in SEQ ID NO: 143). In one embodiment, the GLP-1 peptide or its analogue or its intermediate peptide is G4 (as shown in SEQ ID NO: 142). In one embodiment, the GLP-1 peptide or its analogue or its intermediate peptide is G1, G2 or G3 (as shown in SEQ ID NO: 139-141, respectively).

[0016] In one implementation, n is selected from 2 to 12, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In one implementation, n is selected from 5 to 9. In one implementation, n is 7.

[0017] In one embodiment, the leader peptide comprises a leader peptide core sequence selected from T3, T5, T6, T8, T9, T12, T13, T14, T15, T16, and T19, and the linker peptide comprises a linker peptide core sequence selected from L1, L2, L3, L5, L7, and L8. In another embodiment, the leader peptide comprises a leader peptide core sequence selected from T3, T5, T6, T8, T9, T12, T13, T14, T15, T16, and T19, and the linker peptide comprises a linker peptide core sequence selected from L1, L2, and L8. In yet another embodiment, the leader peptide comprises a leader peptide core sequence selected from T3, T9, T12, T14, T16, and T19, and the linker peptide comprises a linker peptide core sequence selected from L1, L2, L3, L5, L7, and L8. In one embodiment, the leader peptide comprises a leader peptide core sequence selected from T3, T9, T12, T14, T16, and T19, and the linker peptide comprises a linker peptide core sequence selected from L1, L2, and L8. In the above embodiments, the GLP-1 peptide or its analogue or intermediate peptide is selected from G1, G2, and G3. In the above embodiments, n is 5 or 7, particularly 7. In the above embodiments, when the leader peptide core sequence is T3, the N-terminus is LysArg or ArgArg. In the above embodiments, when the leader peptide core sequence is T5, T6, T8, T9, T12, T13, T14, T15, T16, or T19, the N-terminus is AspAspAspAspLys.

[0018] In one embodiment, the polypeptide construct is shown as any one of SEQ ID NO: 1-111. In one embodiment, the polypeptide construct is as shown in SEQ ID NO: 3, 5, 6, 8, 9, 12, 14, 15, 17, 18, 21, 23, 24, 26, 27, 39, 41, 42, 44, 45, 48, 49, 50, 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 69, 70, 73, 74, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, or 111. In one embodiment, the polypeptide construct is as shown in SEQ ID NO: 3, 9, 12, 18, 48, 50, 53, 55, 70, 74, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110.

[0019] In one implementation, T is missing.

[0020] On one hand, the present invention provides a polynucleotide encoding the precursor peptide, linker peptide, or polypeptide construct of the present invention. In one embodiment, the polynucleotide is codon-optimized according to the codon preference of *E. coli*.

[0021] On one hand, the present invention provides a vector comprising the polynucleotides of the present invention. In one embodiment, the vector is a cloning vector or an expression vector. In one embodiment, the vector is a plasmid.

[0022] On one hand, the present invention provides a host cell comprising the polynucleotides of the present invention. On another hand, the present invention provides a host cell comprising the vector of the present invention. On yet another hand, the present invention provides a host cell expressing the polypeptide construct of the present invention. In one embodiment, the host cell is *Escherichia coli*.

[0023] On one hand, the present invention provides a method for preparing GLP-1 peptides or their analogues or intermediate peptides, comprising:

[0024] (1) Express the polypeptide construct of the present invention in Escherichia coli host cells; and

[0025] (2) The expressed polypeptide constructs were treated with enterokinase (EK) (EC 3.4.21.9), Kex2 enzyme (EC 3.4.21.61) and carboxypeptidase B (CpB) (EC 3.4.17.2).

[0026] In one embodiment, step (1) is performed in a shake flask or fermenter. In one embodiment, step (1) is performed using high-density fermentation. In one embodiment, step (1) includes a host cell growth phase and a target product production phase. In one embodiment, the target product production phase includes the addition of an inducer. In one embodiment, the inducer is IPTG.

[0027] In one embodiment, in step (2), EK enzyme is added in an amount not exceeding 10 kU, 9 kU, 8 kU, 7 kU, 6 kU, 5 kU, 4 kU, 3 kU, 2 kU, 1 kU, 0.9 kU, 0.8 kU, 0.7 kU, 0.6 kU, 0.5 kU, 0.4 kU, 0.3 kU, 0.2 kU, or 0.1 kU per gram of the tandem protein (or intermediate peptide) to be cleaved. In one embodiment, Kex2 enzyme is added in an amount not exceeding 20 mg, 19 mg, 18 mg, 17 mg, 16 mg, 15 mg, 14 mg, 13 mg, 12 mg, 11 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, 0.9 mg, 0.8 mg, 0.7 mg, 0.6 mg, 0.5 mg, 0.4 mg, 0.3 mg, 0.2 mg, or 0.1 mg per gram of the tandem protein (or intermediate peptide) to be cleaved. In another embodiment, CpB enzyme is added in an amount not exceeding 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, 0.9 mg, 0.8 mg, 0.7 mg, 0.6 mg, 0.5 mg, 0.4 mg, 0.3 mg, 0.2 mg, or 0.1 mg per gram of the tandem protein (or intermediate peptide) to be cleaved.

[0028] In one implementation, EK enzyme, Kex2 enzyme, and CpB enzyme are added sequentially for treatment.

[0029] In one embodiment, the method of the present invention achieves intermediate peptide yields of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 mg / L or higher. In one embodiment, the intermediate peptide yield is achieved in shake-flask trials. In one embodiment, the shake-flask trials are conducted at scales of 50 mL, 100 mL, 200 mL, 250 mL, 500 mL, 1 L, 2 L, and 4 L. In one embodiment, the yield is achieved without recovery, separation, and / or purification steps. In one embodiment, the yield is achieved after recovery, separation, and / or purification steps.

[0030] In one embodiment, the method of the present invention achieves intermediate peptide yields of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 g / L or higher. In one embodiment, the intermediate peptide yield is achieved in a fermenter pilot-scale. In one embodiment, the fermenter pilot-scale is at a scale of 10 L, 30 L, 50 L, and 100 L. In one embodiment, the fermenter pilot-scale is a fed-batch culture. In one embodiment, the fermenter pilot-scale is a continuous culture. In one embodiment, the fermenter pilot-scale is a batch culture. In one embodiment, the yield is achieved without recovery, separation, and / or purification steps. In one embodiment, the yield is achieved after recovery, separation, and / or purification steps.

[0031] In one embodiment, the method of the present invention achieves an enzymatic digestion efficiency of 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or higher. In one embodiment, the enzymatic digestion efficiency is achieved without a purification step. In another embodiment, the enzymatic digestion efficiency is achieved after a purification step.

[0032] In one embodiment, the method further includes one or more of the following steps: host cell disruption, inclusion body isolation, inclusion body washing, inclusion body dissolution, filtration of the enzymatic digestion solution, isolation of intermediate peptides, and purification of intermediate peptides.

[0033] On the one hand, the present invention provides the use of the precursor peptide of the present invention or the polynucleotide encoding the precursor peptide of the present invention in the preparation of GLP-1 peptide or analogues thereof.

[0034] On the one hand, the present invention provides the use of the linker peptide of the present invention or the polynucleotide encoding the linker peptide of the present invention in the preparation of GLP-1 peptide or analogues thereof.

[0035] On the one hand, the present invention provides the use of the polypeptide construct of the present invention or the polynucleotide encoding the polypeptide construct of the present invention in the preparation of GLP-1 peptide or analogues thereof. Attached Figure Description

[0036] Figure 1: Electrophoresis diagram of shake-flask small-scale induced expression of construct SEQ ID NO: 48. Wherein: lane M is the 180kDa protein marker (Novizan, MP102), lane 1 is the supernatant after induction and lysis of engineered bacteria, and lane 2 is the inclusion body precipitate after induction and lysis of engineered bacteria.

[0037] Figure 2: Electrophoresis diagram of shake-flask small-scale induced expression of construct SEQ ID NO: 78. Wherein: M is the 180kDa protein marker (Novazia, MP102), lane 1 is the supernatant after induction and lysis of engineered bacteria, and lane 2 is the inclusion body precipitate after induction and lysis of engineered bacteria.

[0038] Figure 3: Electrophoresis diagram of shake-flask small-scale induced expression of construct SEQ ID NO: 80. Wherein: M is the 180kDa protein marker (Novizan, MP102), lane 1 is the supernatant after induction and lysis of engineered bacteria, and lane 2 is the inclusion body precipitate after induction and lysis of engineered bacteria.

[0039] Figure 4: HPLC analysis of the construct SEQ ID NO: 48 after shake-flask digestion with EK, Kex2, and CpB. The intermediate peptide eluted at 19.476 min, with a peak area of ​​94.63%.

[0040] Figure 5: HPLC analysis of the construct SEQ ID NO: 78 after shake-flask digestion with EK, Kex2, and CpB. The intermediate peptide eluted at 20.509 min, with a peak area of ​​92.55%.

[0041] Figure 6: HPLC analysis of the construct SEQ ID NO: 80 after triple digestion with EK, Kex2, and CpB in a shake-flask experiment. The intermediate peptide eluted at 20.593 min, with a peak area of ​​91.37%.

[0042] Figure 7: Mass spectrometry analysis of the construct SEQ ID NO: 48 after shake-flask digestion with EK, Kex2, and CpB. The molecular weight of the intermediate peptide was 3173.418, consistent with the theoretical value of 3175.

[0043] Figure 8: Mass spectrometry analysis of the construct SEQ ID NO: 78 after digestion with EK, Kex2, and CpB in a shake-flask experiment. The molecular weight of the intermediate peptide was 3173.419, consistent with the theoretical value of 3175.

[0044] Figure 9: Mass spectrometry analysis of the construct SEQ ID NO: 80 after shake-flask digestion with EK, Kex2, and CpB. The molecular weight of the intermediate peptide was 3173.417, consistent with the theoretical value of 3175.

[0045] Figure 10: HPLC chromatogram of the pilot-scale fermentation of the construct SEQ ID NO: 48 after purification by ion exchange chromatography. The purity of the main peak in the ion exchange chromatography was 97.71%.

[0046] Figure 11: HPLC chromatogram of the construct SEQ ID NO: 78 after pilot-scale fermentation and purification by ion exchange chromatography. The purity of the main peak in ion exchange chromatography was 94.91%.

[0047] Figure 12: HPLC chromatogram of the construct SEQ ID NO: 80 after pilot-scale fermentation and purification by ion exchange chromatography. The purity of the main peak in ion exchange chromatography was 94.67%. Detailed Implementation

[0048] This invention produces intermediate peptides of GLP-1 peptide or its analogues through tandem proteins, and obtains intermediate peptide monomers by cleaving the tandem proteins with Kex2 and CpB.

[0049] This invention enables the production of GLP-1 peptides or their analogues via high-density fermentation. High-density fermentation refers to a technique where the density of microorganisms in a liquid culture medium exceeds 10 times that of conventional methods. Specifically, the cell density of genetically engineered Escherichia coli exceeds 250 g / L.

[0050] The production method of this invention is simple, requires low amounts of enzymes (EK, Kex2, and CpB), and achieves high yield and purity. After fermentation, enzymatic digestion, and chromatography, the intermediate peptide yield is greater than 10 g / L of fermentation broth, and the HPLC purity is greater than 95%.

[0051] Example 1: Design of GLP-1 analog intermediate peptide tandem protein

[0052] The GLP-1 analog intermediate peptides were tandemly repeated 6 or 8 times. The amino acid sequences of the GLP-1 analog intermediate peptides are shown in Table 3. Linker peptides were inserted between the GLP-1 analog intermediate peptides. The N-terminus of the linker peptide contained a Kex2 cleavage site (LysArg or ArgArg), and the C-terminus contained an enterokinase EK cleavage site (AspAspAspAspLys). The core amino acid sequence of the linker peptide is shown in Table 2. A leader peptide was introduced at the N-terminus of the polypeptide chain. The C-terminus of the leader peptide contained a Kex2 cleavage site (LysArg or ArgArg) or an enterokinase EK cleavage site (AspAspAspAspLys). The core amino acid sequence of the leader peptide is shown in Table 1. The amino acid sequence of the designed GLP-1 analog intermediate peptide tandem protein is shown in Table 4.

[0053] Example 2: Construction of recombinant engineered bacteria

[0054] Based on the codon preference of *E. coli*, a nucleotide sequence encoding the tandem protein of Example 1 was designed. The coding sequence was spliced ​​using overlap PCR, and inserted into the vector pET-28a(+) using the NcoI and XhoI sites located at the 5' and 3' ends, respectively, and transformed into *E. coli* strain DH5a (Novizan, catalog number C502). The sequenced-validated plasmid was transformed into *E. coli* strain BL21(DE3) (Novizan, catalog number C504). Single colonies were selected for initial screening based on tandem protein yield.

[0055] Example 3: Small-scale shake-flask test

[0056] The bacterial strain obtained in Example 2 was inoculated into 50 mL of LB liquid medium, and kanamycin monosulfate was added to a concentration of 50 mg / L. The culture was incubated overnight at 37°C with shaking. The next day, the strain was inoculated into 1 L of LB liquid medium, and kanamycin monosulfate was added to a concentration of 50 mg / L. The culture was then incubated at 37°C with shaking. When OD... 600 When the value reached 0.6, IPTG was added to 0.5 mM to induce tandem protein expression. After 12 hours, the bacterial cells were collected by centrifugation and weighed.

[0057] Weigh 2g of bacterial cells and add 40mL of disruption buffer (20mM Tris, 200mM NaCl, 1mM EDTA·2Na, pH 7.5). Mix thoroughly by pipetting in a 50mL centrifuge tube (Novizan, catalog number TCF00150). Disrupt the cells using an ultrasonic homogenizer (Nanjing Xianou Instrument Manufacturing Co., Ltd., model ATPIO-1000D) under ice bath conditions. Use an amplitude transformer Φ6, power 30%, for 3-second intervals with 2-second intervals, for a total duration of 20 minutes. Centrifuge the disruption solution at 10000-13000g for 5-10 minutes, then transfer the supernatant to a new 50mL centrifuge tube. Add 40mL of disruption buffer to the precipitate to form an inclusion body suspension. Take 80 μL of the supernatant and suspension respectively, and add 20 μL of 5×SDS-PAGE protein loading buffer (Shanghai Yisheng, 20315ES05) to each. Incubate at 100℃ for 5 min to allow the protein to denature fully. After cooling to room temperature, take 10 μL of each and add 4%-20% SmartPAGE buffer. TM The protein gel (Tiandi Renhe, SLE020) was loaded into the sample wells. In another sample well, 10 μL of 180 kDa protein marker (Novazia, MP102) was added, resulting in 1.0 μg of protein per band. Electrophoresis was performed at 160 V, and staining and destaining were performed using an M-Blot rapid transfer instrument (Zhongke Tongyi, TY-201-01) (see Figures 1-3). The gel images were analyzed using ImageJ software (National Institutes of Health, version 1.54g) to obtain the IntDen grayscale values ​​of the protein bands.

[0058] Disrupt the remaining bacterial cells using the same procedure described above. Then, add the same volume of washing buffer (20 mM Tris, 1 mM EDTA·2Na, pH 7.5) as the disruption buffer to the inclusion body precipitate, mix thoroughly by pipetting, and centrifuge at 10000-13000g for 5-10 minutes. Discard the supernatant. Repeat this washing process three times. Resuspend the inclusion body precipitate to 40 g / L with dissolution buffer (20 mM Tris, 8 M urea, pH 7.5). Slowly dilute the urea concentration to 2 M with dilution buffer (20 mM Tris, pH 7.5) while continuously stirring, and dissolve the inclusion body precipitate for 2-6 hours.

[0059] Add 2-3 kU EK (Novazia, catalog number RM1025), 3-5 mg Kex2 (Novazia, catalog number JE1016L), and 2-3 mg CpB (Novazia, catalog number JE1002L) to a buffer containing 1 g of renatured tandem protein. Incubate at 30°C with shaking at 50-100 rpm for 6 hours. Samples are then analyzed by HPLC (Thermo Fisher Scientific Vanquish-Gore high-performance liquid chromatograph) and mass spectrometry (Orbittrap XL classic combination mass spectrometer). Based on the HPLC chromatogram, the percentage of the main peak is calculated by peak area, and the amount of protein in the main peak is measured using the external standard method.

[0060] HPLC conditions

[0061] Column: ChromCore 300C4-T 3μm, 4.6×150mm

[0062] Column temperature: 35℃

[0063] Injection volume: 5 μL

[0064] Detection wavelength: 214nm

[0065] Flow rate: 0.5 mL / min

[0066] Mobile phase A: 0.1% trifluoroacetic acid (20% acetonitrile)

[0067] Mobile phase B: 0.1% trifluoroacetic acid (acetonitrile)

[0068] Gradient elution:

[0069] Mass spectrometry conditions

[0070] Vaporization temperature: 300℃

[0071] Voltage: 4kV

[0072] Detector: FTMS

[0073] Scanning method: Full scan

[0074] Mobile phase A: 0.1% formic acid (water)

[0075] Mobile phase B: 0.1% formic acid (acetonitrile)

[0076] The following evaluation was conducted (results are shown in Table 5):

[0077] 1) The measured yield of tandem protein (mg / L) = [IntDen gray value of tandem protein inclusion body band / IntDen gray value of protein marker band × 1.0 (μg) × 40 (mL) × cell weight per L of liquid culture medium (g)] / [10 (μL) × 0.8 * 2 (g)];

[0078] 2) The calculated percentage of intermediate peptide molecular weight (%) = [molecular weight of intermediate peptide (kDa) × (n+1) / molecular weight of tandem protein (kDa)] × 100%;

[0079] 3) Estimated yield of intermediate peptide (mg / L) = Measured yield of tandem protein (mg / L) × Calculated percentage of intermediate peptide molecular weight (%);

[0080] 4) The measured yield of intermediate peptide (mg / L) = [amount of protein obtained by HPLC external standard method (μg) / sample volume of enzyme digestion solution used for HPLC (μL) × total volume of enzyme digestion solution (mL)] / 1 (L);

[0081] 5) The calculated value of the enzymatic conversion rate of the intermediate peptide (%) = [the measured value of the yield of the intermediate peptide (mg / L) / the estimated value of the yield of the intermediate peptide (mg / L)] × 100%.

[0082] The results showed that: 1) After tandem protein digestion with EK, Kex2, and CpB, the main peak percentage ranged from 80% to 94%, with most engineered bacteria showing a main peak percentage between 89% and 93% (see Table 5). 2) Comparing the estimated and measured yields of intermediate peptides, the digestion conversion rates of most constructs were between 89% and 93% (see Table 5). 3) HPLC chromatograms (see Figures 4-6) showed that the intermediate peptide eluted at approximately 20 minutes, accounting for 90-95% of the peak; mass spectrometry (see Figures 7-9) showed that the molecular weight of the intermediate peptide was approximately 3173.42, consistent with the theoretical calculation of 3175.

[0083] Example 4: Pilot-scale test in a fermenter

[0084] Engineered bacteria that achieved an actual yield of intermediate peptides above 165 mg / L and exhibited a main enzyme digestion peak percentage above 90% in shake-flask small-scale trials were used for pilot-scale fermentation. The first-stage seed culture consisted of 50 mL LB medium, and the second-stage seed culture consisted of 1 L LB medium. After inoculating the engineered bacteria into the first-stage seed culture, the cultures were incubated at 37°C with shaking until OD reached [value missing]. 600 The value reached 2. Inoculate with grade 2 seed culture and incubate at 37°C with shaking until the OD value reaches 2. 600The pH value reached 2. The inoculation was carried out in a stainless steel fermenter (Bailun Biotechnology, model BLB10-50SJ-2). The pilot-scale operation was as follows: Weigh 266g KH₂PO₄, 80g (NH₄)₂HPO₄, 38g citric acid monohydrate, 24g MgSO₄·7H₂O, 1.2g ferric citrate, 0.3g manganese chloride tetrahydrate, 0.16g zinc acetate dihydrate, 0.06g boric acid, 0.05g sodium molybdate dihydrate, 0.05g cobalt chloride hexahydrate, 0.03g copper chloride dihydrate, 0.168g EDTA·2Na, and 2041mL defoamer. Dissolve these in 16L DDW and sterilize in the fermenter. Weigh 7000g anhydrous glucose and 200g magnesium sulfate heptahydrate, dissolve and dilute to 10L DDW as the feed carbon source. Use 28% ammonia water as a pH adjuster. Inoculate 1 L of LB medium, a glucose stock solution containing 300 g of anhydrous glucose, and 1 g of kanamycin monosulfate into the fermenter. Set the temperature to 37°C, maintain the pH at 7.0 using 28% ammonia, aerate at 1-2 VVM, and pressurize the fermenter to 0.05-0.1 MPa to begin fermentation. Once the glucose in the substrate is depleted, replenish the carbon source using a peristaltic pump as shown in the table below. During the replenishment process, add pure oxygen to maintain dissolved oxygen >20%. When OD... 600 When the concentration reaches 120, add 1M IPTG stock solution to a final concentration of 0.2mM for induction. After 8-12 hours, centrifuge to collect the bacterial cells and weigh the wet weight of the cells.

[0085] Feeding rate:

[0086] Add 10L of disruption buffer (20mM Tris, 200mM NaCl, 1mM EDTA·2Na, pH 7.5) to each kg of bacterial cells, mix well, filter through a 300-mesh filter to remove impurities, and use a pilot-scale high-pressure homogenizer (Shanghai Duoning Biotechnology Co., Ltd., model AH-PILOT) at 700-750 bar to cycle the homogenate 3-5 times until the solution becomes relatively clear and free of any flocculent matter. Centrifuge the lysate at 10000-13000g for 5-10 minutes, and collect the inclusion body precipitate and supernatant separately for electrophoresis. Measure the tandem protein content by grayscale scanning, and the result is 15-22g / L.

[0087] Add the same volume of washing buffer (20 mM Tris, 1 mM EDTA·2Na, pH 7.5) as the disruption buffer to the inclusion body precipitate. Mix well using a top-mounted electronic stirrer (DLAB OS20-Pro). Centrifuge at 10000-13000g for 5-10 minutes and discard the supernatant. Repeat this washing process 3 times. Resuspend the inclusion body precipitate to 40 g / L with dissolution buffer (20 mM Tris, 8 M urea, pH 7.5). Slowly dilute the urea concentration to 2 M with dilution buffer (20 mM Tris, pH 7.5) while continuously stirring, and dissolve the inclusion body precipitate for 2-6 hours.

[0088] Enzyme digestion was performed as described in Example 3.

[0089] The sample was filtered sequentially using 1μm and 0.45μm filter cartridges to remove insoluble matter, followed by ion exchange chromatography. Before sample loading, the chromatography column (Chutian Technology, model QFF) was equilibrated with 3-5 column volumes of equilibration buffer (20mM Tris, 1M NaCl, pH 10.5). After sample loading, the column was washed with 2-5 column volumes of equilibration buffer. Following pre-washing with pre-wash buffer (20mM Tris, 500mM NaCl, pH 10.5), elution was performed using pure water as the elution buffer. (Based on OD...) 280nm The absorbance value was used to collect the main peak of the eluent, and the elution peak was detected by HPLC.

[0090] The amount of protein in the elution peak was measured by HPLC external standard method, and the yield was calculated as follows:

[0091] The yield of the purified intermediate peptide (g / L) = [amount of protein obtained by HPLC external standard method (μg) / volume of elution peak sample used for HPLC (μL) × total volume of elution peak (L)] / fermentation tank volume (L).

[0092] As shown in Figure 10, the intermediate peptide purity of the main elution peak of SEQ ID NO: 48 by ion exchange chromatography was 97.71%, and the yield was 9.23 g / L; as shown in Figure 11, the intermediate peptide purity of the main elution peak of SEQ ID NO: 78 by ion exchange chromatography was 94.91%, and the yield was 10.68 g / L; as shown in Figure 12, the intermediate peptide purity of the main elution peak of SEQ ID NO: 80 by ion exchange chromatography was 94.67%, and the yield was 12.26 g / L.

[0093] Table 1: Core Sequence of Leader Peptide

[0094] Table 2: Core Sequence of Linker Peptides

[0095] Table 3: Amino acid sequences of GLP-1 peptide and its intermediate peptides

[0096] Table 4: Sequences of tandem protein constructs (Construction number = SEQ ID NO)

[0097] Table 5: Results of the shake-flask trial

[0098] Intermediate peptide yield measurements: 151-205 mg / L rated ++++, 121-150 mg / L rated +++, 81-120 mg / L rated ++, and 55-80 mg / L rated +.

[0099] The percentage of the main enzyme digestion peak is rated as follows: 89%-94% is rated as ***, 85%-88% is rated as **, and 80%-84% is rated as *.

Claims

1. A precursor peptide having the general formula: Met–precursor peptide core sequence–LysArg or ArgArg or AspAspAspAspLys, wherein the precursor peptide core sequence is selected from T1-T19, preferably T3, T5, T6, T8, T9, T10, T11, T12, T13, T14, T15, T16, T17, T18 and T19, more preferably T3, T5, T6, T8, T9, T12, T13, T14, T15, T16 and T19, most preferably T3, T9, T12, T14, T16 and T19, optionally… When the N-terminus is LysArg or ArgArg, the core sequence of the leader peptide is T1-T4, with T3 being the most preferred. When the N-terminus is AspAspAspAspLys, the core sequence of the leader peptide is T5-19, preferably T5, T6, T8, T9, T10, T11, T12, T13, T14, T15, T16, T17, T18 and T19, with T9, T12, T14, T16 and T19 being the most preferred.

2. A linker peptide having the general formula: LysArg or ArgArg – linker peptide core sequence – AspAspAspAspLys, wherein the linker peptide core sequence is selected from L1-L9, preferably L1, L2, L3, L5, L7 and L8, and most preferably L1, L2 and L8.

3. A polypeptide construct having the general formula: T–G–(L–G) n ,in, T is the precursor peptide according to claim 1. L is the linker peptide according to claim 2. G is a GLP-1 peptide or an analogue as shown in G5, preferably G4, such as G1, G2 or G3. n is a natural number, preferably 2-12, further preferably 5-9, and most preferably 7. Preferred, The polypeptide construct is as shown in any of SEQ ID NO: 1-111, more preferably as shown in SEQ ID NO:

1. NO: 3, 5, 6, 8, 9, 12, 14, 15, 17, 18, 21, 23, 24, 26, 27, 39, 41, 42, 44, 45, 48, 49, 50, 53, 54, 55, 56, 57, 58, 59, 62, 63, 64, 65, 69, 70, 73, 74, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 or 111, with the most preferred option as shown in SEQ ID. NO: 3, 9, 12, 18, 48, 50, 53, 55, 70, 74, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 as shown. Optional T is missing.

4. A method for preparing intermediate peptides of GLP-1 peptide or analogues thereof, comprising: (1) Express the polypeptide construct according to claim 3 in Escherichia coli host cells; and (2) The expressed polypeptide constructs were treated with enterokinase (EK) (EC 3.4.21.9), Kex2 enzyme (EC 3.4.21.61) and carboxypeptidase B (CpB) (EC 3.4.17.2).

5. Use of the precursor peptide according to claim 1 or the polynucleotide encoding the precursor peptide according to claim 1 in the preparation of GLP-1 peptide or analogues thereof.

6. Use of the linker peptide of claim 2 or the polynucleotide encoding the linker peptide of claim 2 in the preparation of GLP-1 peptide or analogues thereof.

7. Use of the polypeptide construct according to claim 3 or the polynucleotide encoding the polypeptide construct according to claim 3 in the preparation of GLP-1 peptide or analogues thereof.

8. A polynucleotide encoding the polypeptide construct according to claim 3, preferably codon-optimized according to Escherichia coli codon preferences.

9. A vector comprising the polynucleotide according to claim 8, optionally, the vector being an expression vector.

10. A host cell comprising the polynucleotide of claim 8, the vector of claim 9, or expressing the polypeptide construct of claim 3, preferably, the host cell being *Escherichia coli*.

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