CN1061373C - Method for preparing monomer insulin - Google Patents

Abstract

The present invention has the contents that a monomer insulin precursor (MIP) is excreted and expressed by an enzyme mother system, and the enzymatic degradation and the peptide conversion of trypsinase for MIP are carried out to obtain b-chain carboxyl terminal pentapeptide-removed or tetrapeptide-removed insulin (DPI or DTI). The recombined monomer short-acting insulin is like natural insulin to not have mutant residues.

Description

The preparation method of monomer insulin

The present invention is the preparation method of monomer insulin.Regular Insulin is the specifics of treatment diabetes, and it is used for clinical existing more than 70 year.But one of present clinical subject matter with insulin preparation, be control patient blood sugar the time phase aspect, the function that can not imitate physiology Regular Insulin.The normal people, dining back 30-60 minute, blood insulin concentration reaches the peak, returns to the base concentration level after 4-5 hour, consistent when this has meal back blood sugar with the people (Schade, D.S., et al., Diabetes Care, 1980,3:9).And behind the subcutaneous injection insulin preparation, blood insulin concentration slowly rises, and only reaches lower peak value after 90-120 minute, can not reduce (Home fast again afterwards, P.D., et al., Metabolism, 1981,30:439), thereby make patient's blood sugar be in non-physiological period state.Its major cause is because the form of the insulin hexamer aggressiveness of drug concentration exists (Blundall, T., et al., Adv.Protein Chem., 1972,26:279; Drejer, K., Diabetes/Metabolism Reviews, 1992,8:259), and Regular Insulin effect in vivo at first is with monomeric form receptors bind single-minded with it, finally shows its physiological function (Blundall, T., etal., Adv.Protein Chem., 1972,26:279; Insulin Research Group, AcademiaSinica, Sci.Sin., 1974,17:779).Behind the medical injection Regular Insulin, change dimer into, become monomer again, need the long time, cause effect slowly by six aggressiveness.If the Regular Insulin of injection is monomer insulin, then can directly plays a role, thereby overcome General Medicine insulin action shortcoming slowly.Therefore, obtaining to can be used for clinical monomer quick-acting insulin, is one of major objective of present insulin protein engineering research.The method of acquisition monomer insulin commonly used is to pass through site-directed point mutation at present, to the dimer formation face of insulin molecule introduce have electric charge or side chain very big amino-acid residue, to hinder dimeric formation, to obtain monomer insulin (Brange, J., et al., Nature, 1988,333:679; Kang, S., et al., Lancet, 1990,335:303; Liu Bin etc., Acta Biochimica et Biophysica Sinica, 1996,38:245).The monomer insulin that is obtained all contain the sudden change residue (Wang Qiongqing etc., biological chemistry and biophysics are made progress, 1996,23:402).

There is (Chinese Academy of Sciences's Shanghai biological chemistry Regular Insulin group in B chain carboxylic end des-pentapeptide-insulin (DPI) with monomeric form, Chinese science, 1976 (4), 429), and keep higher insulin activity (InsulinResearch Group, Academia Sinica, Sci.Sin., 1974,17:779).For obtaining can be used for clinical reorganization DPI, we are with Regular Insulin strand precursor (PIP) gene (Zhang, You-Shang, etal., Science in China (Series C), 1996,39:225) the Lys codon of B29 position is changed into the Thr codon, obtains containing the plasmid of MIP gene and carry out secreting, expressing in wine brewing ferment yeast.And then with the MIP of purifying and the tripeptides Gly-Phe-Phe or the tetrapeptide Gly-Phe-Phe-Tyr (OBut) of chemosynthesis ₂Be substrate, change peptide by the trypsinase enzymatic and obtain reorganization DPI or DTI.The reactive site of MIP and tripeptides or tetrapeptide is the carboxyl of MIP B22 position Arg.Reorganization DPI and DTI are the natural monomer quick-acting insulins that does not contain the residue that suddenlys change.

The present invention is a secreting, expressing monomeric insulin precursor (MIP) and change the monomer insulin (DPI, DTI and other monomer insulin) that peptide obtains by enzymatic in yeast, and its feature is as follows:

A. use the site-directed point mutation method, the B29Lys codon in Regular Insulin strand precursor (PIP) gene changed into arbitrary amino acid whose codon beyond Lys and the Arg, obtain MIP expression plasmid pZW[B29Thr] PIP.

B. use plasmid pZW[B29Thr] PIP transformed saccharomyces cerevisiae Saccharomycescerevisiae (XV-700-6B) and obtain engineering strain YS98.

C. secreting, expressing product MIP is converted into DPI, DTI and other monomer insulin by enzymatic commentaries on classics peptide after separation and purification.

Technical characterictic of the present invention is described specifically in following embodiment.

Explanation of nouns:

MIP: monomeric insulin precursor

PIP: Regular Insulin strand precursor

DTI:B chain carboxylic end removes tetrapeptide Regular Insulin

DPI:B chain carboxylic end des-pentapeptide-insulin

DOI:B chain carboxylic end removes octapeptide Regular Insulin

Description of drawings of the present invention is as follows:

The mensuration of Fig. 1 mutant nucleotide sequence

The visible AGG of arrow indication has sported ACC

The Sephadex G-50 of Fig. 2 MIP (2.5 * 160cm) wash-out collection of illustrative plates

Dash area is exempted from the active zone for putting among the figure.

Fig. 3 pH8.3 polyacrylamide discontinuous electro-phoresis

Be followed successively by from left to right:

DOI (standard), DPI (standard), DPI, through the MIP of gel-filtration and HPLC purifying,

MIP through the gel-filtration purifying

The RP-HPLC separation graph of Fig. 4 MIP

Column:C8(Beckman),1.0×25cm

Buffer A:0.1％TFA

Buffer B:70% acetonitrile, 0.08%TFA

Gradient:40-55％B/10-40min

Flow rate:2ml/min

A ₂₃₀

The HPLC of Fig. 5 MIP (separating through HPLC) identifies figure

Column:C8(Beckman),1.0×25cm

Buffer A：0.1％TFA

Buffer B:70% acetonitrile, 0.08%TFA

Gradient:40-55％B/10-40min

Flow rate:2ml/min

A ₂₃₀

The mass spectrum of Fig. 6 MIP is identified

MIP MW 5931.72 is determined as 5932.0

5954 is MIP-Na, and 5970 is MIP-K

Fig. 7 transpeptidation reaction mixture HPLC separation graph

Peak 1 DOI, peak 2 DPI

Column:C8(Beckman),1.0×25cm

Buffer A:0.1％TFA

Buffer B:70% acetonitrile, 0.08%TFA

Gradient:40-55％B/10-40min

Flow rate:2ml/min

A ₂₃₀

The mass spectrum of Fig. 8 DPI is identified

DPI MW 5214.85 is determined as 5216.0

5255 is DPI-K

Fig. 9 pork insulin and reorganization DPI are right ¹²⁵I-Regular Insulin and the membrane-bound restraining effect of people's placenta cells

Solid line is reorganization DPI among the figure, and dotted line is a pork insulin.

Reorganization DPI combination rate is 72% of a pork insulin.

Figure 10 MIP is converted into the RP-HPLC separation graph of DTI

Column:C8(Beckman),1.0×25cm

Buffer A:0.1％TFA

Buffer B:0.08％TFA,70％CH ₃CN

Gradient:40-50％B/10-30min,50-75％B/30-55％B

Flow rate:2ml/min

A ₂₃₀

The RP-HPLC of Figure 11 DTI identifies figure

Column:C8(Beckman),1.0×25cm

Buffer A:0.1％TFA

Buffer B:0.08％TFA,70％CH ₃CN

Gradient:40-50％B/10-30min,50-75％B/30-55％B

Flow rate:2ml/min

A ₂₃₀

Figure 12 PAGE electrophorogram

Be followed successively by Regular Insulin from left to right, MIP, DTI, DTI (OBut) ₂

The mass spectrum of Figure 13 DTI is identified

DTI MW is 5380.02, is determined as 5380.0

Embodiment 1. site-directed point mutations

Mutant primer: be Thr codon (ACC) with the Lys codon mutation during 5 ' TTC TAC ACT CCT ACC GCT GCT AAG GG 3 ' design, because similar Lys of the wetting ability of Thr and the Thr tRNA abundance in yeast cell is higher.After the jump reaction product transformed JM105, the picking clone identified mutant (Fig. 1) with the dna sequencing method, obtains the expression plasmid pZW[B29Thr of MIP] PIP.

The secreting, expressing of embodiment 2.MIP in yeast

With plasmid pZW[B29Thr] PIP transformed saccharomyces cerevisiae Saccharomyces cerevisiaeXV700-6B is built into engineering bacteria YS98.Get YS98 and be inoculated in the 5mlYPD nutrient solution, in 30 ℃, grow overnight is transferred in the 50mlYPD nutrient solution, after about 20 hours of the continued growth, changed the 10L fermentor cultivation over to three days (Zhang, You-Shang, et al., Science in China (Series C), 1996,39:225).Measure the YS98 bacterial strain in shaking bottle and 10L fermentor tank with the Regular Insulin radioimmunoassay, the expression level of MIP is respectively 6mg/ and rises and the 38mg/ liter.

Separation and purification and the evaluation of embodiment 3. expression product MIP

The centrifugal thalline of removing is got fermented liquid and is added trichloroacetic acid precipitation protein in 0 ℃, and centrifugal collecting precipitation behind the static 30min is to 7% acetate dialysed overnight.Centrifugal, get Sephadex G-50 post on the supernatant, with 7% acetate wash-out, the peak (Fig. 2) of collection and PIP corresponding position, lyophilize.Separate once lyophilize again with above-mentioned SephadexG-50 post.Gained [B29Thr] MIP is accredited as single band (Fig. 3) with polyacrylamide gel electrophoresis.With above-mentioned separation and purification program, from 1 liter of fermented liquid, can get MIP 34mg.

The learn from else's experience MIP of Sephadex G-50 column purification further uses HPLC purifying (Fig. 4), obtains high purity MIP (Fig. 3, Fig. 5) after collecting the main peak freeze-drying.Mass spectrometry results, molecular weight conform to theoretical value (Fig. 6).This step rate of recovery is 26.7%.

It is reorganization B chain carboxylic end des-pentapeptide-insulin that embodiment 4. usefulness enzymatics change peptide method conversion MIP

The MIP that gets quantitative HPLC purifying is dissolved in DMSO, 1, in the mixed solution of 4-butyleneglycol/water, adds excessive 20 times tripeptides HClGly-Phe-Phe, transfer pH to 8 with the M-methylmorpholine, add trypsin MIP: enzyme=5: 1, w/w), in 37 ℃ of reactions 20 hours, add the acetone termination reaction.The HPLC analysis revealed has 62% MIP to change DPI into, and 38% MIP is for removing B chain carboxylic end octapeptide Regular Insulin (DOI) (Fig. 7).The DPI that gained is pure, PAGE electrophoresis homogeneous (Fig. 3).Mass spectroscopy and aminoacid sequence N end 1-24, C end 1-3 measurement result show gained reorganization DPI molecule complete, correct (Fig. 8).The rate of recovery of changeing peptide is 16.7%, still can carry out enzymatic after DOI reclaims and change peptide, so the ultimate yield of DPI also can further improve.

The biologos of embodiment 5.B chain carboxylic end des-pentapeptide-insulin and the evaluation of monomer performance

Reorganization DPI and people's placenta cells film insulin binding ability are as shown in Figure 9, calculate with combination rate 50%, the binding ability of DPI and acceptor is 72% (Feng Youmin etc., Acta Biochimica et Biophysica Sinica, 1982 of natural pork insulin, 14:137), (Insulin Research Group, Academia Sinica, Sci.Sin. conform to the preamble report, 1974,17:779).

With sxemiquantitative small white mouse convulsions method, it is as shown in table 1 to measure the interior vigor of reorganization DPI body.

The monomer behavior of reorganization DPI conform to DPI (Shanghai Inst. of Biochemistry, Chinese Academy of Sciences's Regular Insulin group, Chinese science, 1976, (4): 429) with the acquisition of enzymolysis or enzymatic synthesis method.

It is that B chain carboxylic end tetrapeptide Regular Insulin is removed in reorganization that embodiment 6. usefulness enzymatics change peptide method conversion MIP

The MIP that gets the 3mgHPLC purifying is dissolved in DMSO/1, among the mixed solution 200 μ l of 4-butyleneglycol/water=15: 70: 15, adds excessive 50 times GFFY (OBut) ₂Transfer pH to 7.0 with the N-methylmorpholine, add trypsinase MIP: enzyme=5: 1, w/w), in 30 ℃ of reaction 20hr, add 80 μ l acetic acid termination reactions, reaction solution divides leave away enzyme and small molecules through Sephadex G-50, lyophilized powder with the acetone dehydration after, with TFA at room temperature reaction 1hr to remove the tert-butyl ester, use HPLC purifying (Figure 10) then, there is 70% MIP to be converted into DTI, gets the pure DTI (Figure 11) of 0.55mgHPLC, PAGE electrophoresis homogeneous (Figure 12), mass spectroscopy shows molecular weight correct (Figure 13), total yield (based on MIP) 20.2%.The Regular Insulin biologos of DTI: vigor is 22U/mg in the full quantitative assay reorganization of small white mouse convulsions method (by British Pharmacopoeia) the DTI body.

Injected dose (ug)	Reorganization DPI	Pork insulin
			0.5	1/10	2/10
1.0	9/10	9/10

Table 1. small white mouse convulsions method is measured vigor in the reorganization DPI body (x/10 represent each dosage make a call to 10 small white mouses and have x only to produce the reaction of fainting from fear)

Claims (3)

1, the preparation method of monomer insulin, with monomeric insulin precursor (monomeric insulinprecursor MIP) obtains B chain carboxylic end with tripeptides or tetrapeptide transpeptidation reaction and removes pentapeptide (DPI) or remove tetrapeptide Regular Insulin (DTI), it is characterized in that:

The tripeptides of described DPI changes the peptide condition: with MIP and Gly-Phe-Phe is substrate, is dissolved in DMSO/1,4-butyleneglycol/water mixed liquid, and PH7.0-8.5 uses trypsinase catalysis;

The tetrapeptide of described DTI changes the peptide condition: MIP and Gly-Phe-Phe-Tyr (Obut) ₂Be substrate, be dissolved in DMSO/1,4-butyleneglycol/water mixed liquid, PH7.0-8.0 uses trypsinase catalysis.

2, the preparation method of monomer insulin according to claim 1, it is characterized in that described MIP is characterised in that the peptide section that connects B22 and A1, do not have other Lys or Arg amino acid except that the basic aminoacids of C end, the reactive site of MIP and tripeptides or tetrapeptide is the carboxyl of MIP B22 position Arg.

3, the preparation method of monomer insulin according to claim 2, it is characterized in that described MIP expresses in Yeast system, expression plasmid is pZW[B29 Thr] PIP, obtain engineering strain YS98 (CGMCCNo.0340) with its transformed saccharomyces cerevisiae Saccharomyces cerevisiae (XV-700-6B).

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