CN1069649C

CN1069649C - [Beta-9 glutamic acid, beta-10 aspartic acid] human insulin

Info

Publication number: CN1069649C
Application number: CN96116230A
Authority: CN
Inventors: 冯佑民; 梁镇和; 唐月华; 刘滨; 张友尚
Original assignee: Shanghai Institute of Biochemistry
Current assignee: Shanghai Institute of Biochemistry
Priority date: 1996-01-25
Filing date: 1996-01-25
Publication date: 2001-08-15
Anticipated expiration: 2016-01-25
Also published as: CN1155548A

Abstract

The present invention relates to human insulin-[B9Glu, B10Asp] human insulin obtained by a protein engineering technology, which is characterized in that both the 9th position and 10th position in the B chain are acidic amino acids, the acceptor combining capacity of the [B9Glu, B10Asp] human insulin is 34.4% of that of pig's insulin, the bioactivity in vivo is basically same with that of natural insulin. The present invention is helpful to be used as a quick-acting insulin monomer in clinical application.

Description

[B9 L-glutamic acid, B10 Aspartic Acid] insulin human

As the specifics of treatment diabetes, Regular Insulin is applied to clinical existing more than 70 year, is the protein drug of consumption maximum clinically.The physiological function of Regular Insulin keeps the normal glucose level of people at what.Usually, blood insulin peaks in 30-60 minute after meal, returns to basal level in 120-180 minute, and is harmonious with the blood sugar peak after having meal.The insulin zinc preparation that contains that uses clinically mainly exists with polymer forms such as hexasomics at present, and Regular Insulin is to show its physiological function with monomeric form.Therefore, be expelled to and be dissociated into monomer gradually after subcutaneous, the peak just appears in 120 minutes blood, and this peak value lasted till 180-240 minute always, caused in patient's for some time internal cause blood insulin concentration to cross low and the hyperglycemia symptom occurred, showed as hypoglycemia (Brange J subsequently again because of blood insulin concentration is too high, et al., Diabetes Care, 1990,13:923).In order to improve this situation, improve the Regular Insulin performance, become (Brange J, et.al., Nature, 1988, the 333:679 of pressing for of clinical treatment with the monomer insulin preparation that obtains the rapid lowering blood glucose level of energy; Brange J, et al., Cur Opin Struct Biol, 1991,934).

Participation effects such as the hydrophobic surface of forming by B12Val, B24 and B25Phe, B16 and B26Tyr etc. when insulin monomer is polymerized to disome, B chain carboxylic end antiparallel and some other hydrogen bond.Three disomes rely on polarity and non-polar action, and coordinate bond forms hexasomic.Therefore, change participation and form of the formation of the amino-acid residue of disome, promptly might obtain monomeric insulin analog with the obstruction disome.

The three-dimensional structural analysis of Regular Insulin shows, B9Ser is positioned at and forms the dimeric hydrophobic surface of Regular Insulin (Baker EN, et al., Phi Trans Roy Soc London B., 1988,319:369), based on this, (Nature, 1988 such as Brange, 333:679) Ser with B9 changes Asp into, repelling dimeric formation, obtain the Regular Insulin that under drug concentration, exists with monomeric form, become Semilente Insulin (the Kang S of clinical needs, et al., Lancet, 1990,335:303), this laboratory replaces B9Ser with Glu, has obtained similar result.But their biologos obviously reduces, and is about 1/2nd of natural insulin.In addition, this laboratory replaces B10His to obtain high vigor [B10Asp] insulin human with Asp.(Biochem Biophys Res Comm such as Burke, 1990,173:982) compared insulin analog a series of and the B10Asp combination, after discovery B10His is replaced by Asp, can generally improve the biologos of corresponding insulin analog with chemical synthesis process.

Based on above result, the present invention utilizes protein engineering method to prepare monomeric insulin analog--[B9 X, B10 Y] insulin human, and wherein X, Y are acidic amino acid and (are embodied as [B9Glu, B10Asp] insulin human; Contain pork insulin precursor (porcine insulinprecursor, PIP) the yeast strain Saccharomycescerevi siae YS92/pLL[B9E of the expressive plasmid of mutator gene, B10D] PIP (XV-700-6B) (CCTCC NO.M95042) has been deposited in Chinese typical culture collection center (Chinese Wuhan Wuhan University in the school).[B9Glu, B10Asp] insulin human's receptor binding capacity is 34.4% of a pork insulin, and biologos is then basic identical with natural insulin in the body.Therefore, the present invention is expected to be applied to as monomer quick-acting insulin clinical.

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

Accompanying drawing of the present invention makes the following instructions:

Fig. 1. the sudden change of pork insulin precursor (PIP) gene.

Fig. 2. the sudden change insulin B chain changes the dna sequence chromatogram in zone.

Fig. 3. expression product is at Sephadex G-50 post (the chromatography collection of illustrative plates on 2.6 * 160cm).

Peak 3 is [B9Glu, B10Asp] PIP.

Fig. 4. polyacrylamide gel electrophoresis (pH8.3) collection of illustrative plates.

It [B9Glu, B10Asp] PIP (A) of Fig. 4 (a) .Sephadex G-50 purifying and further through [B9Glu, the B10Asp] PIP (B) of HPLC purifying.

Fig. 4 (b) .B30Thr (Bu ^t)-OBu ^t[B9Glu, B10Asp] insulin human (A); [B9Glu, B10Asp] insulin human (B); Contrast (C): top band is B30Thr (Bu ^t)-OBu ^tThe insulin human is a pork insulin by following band.

Fig. 5. Regular Insulin HPLC collection of illustrative plates.

Fig. 5 (a). [B9Glu, B10Asp] PIP of purifying.

Fig. 5 (b) [B9Glu, B10Asp] insulin human.

Fig. 6. the HPLC collection of illustrative plates of transpeptidation reaction mixture.

Fig. 7 .[B9Glu, B10Asp] insulin human (o-o) and pork insulin (x-x) and people's placenta cells film insulin binding curve.

The sudden change of embodiment 1. pork insulin precursor (PIP) genes

Clone's step of employing standard (Sambrook J, et al. (eds.) Molecularcloning:A laboratory manual, 1989,2nd ed., Cold SpringHarbor Laboratory, New York), the principle of sudden change and step are as shown in Figure 1.Intestinal bacteria JM103 grows in the 2YT substratum, transforms with the calcium chloride method.Plasmid pVT102-U is Thierry Vernet (Biotechnology Research Institute, Montreal) give, Saccharomyces cerevisiae XV700-6B (Leu2, Ura3, Pep4) be Michael Smith (University of British Columbia, Vancouver) give, in the YPD substratum, grow, transform with the Lithium Acetate method.Transformed yeast cells is with selecting substratum (2% agar, 0.75% yeast nitrogenous base, the amino acid of 2% glucose and each 100 μ g/ml) to select.

Mutant primer is 5 '-CTTGTGCGGTGAAGACTTGGTTGAGGC-3 ', wherein B9Ser and B10His codon TCC and CAC, codon GAA and the GAC with 6lu and Asp replaces respectively in primer.Contain the uridylic single stranded DNA by (Sambrook J such as Sambrook, et al. (eds.Molecular cloning:A laboratory manual, 1989,2nd ed., ColdSpring Harbor Laboratory, New York) method preparation, (Lee is equality also, biotechnology journal, 1987 with reference to Lee etc., 3:90) method is carried out the positional mutation of gene with breach double-stranded DNA method.

Press Sanger etc. (Sanger FG, et al.Proc Natl Acad Sci USA, 1977,47:5463) method is measured the DNA sequence of muton, the result shows that (Fig. 2) correctly takes place in sudden change.And it is transformed into yeast.The yeast pearl Saccharomyces cerevisiae YS92/pLL[B9E that contains the expressive plasmid of mutator gene, B10D] PIP (XV-700-6B) (CCTCC NO:M95042) has been deposited in Chinese typical culture collection center (Chinese Wuhan Wuhan University in the school).

Embodiment 2.[B9Glu, B10ASp] secreting, expressing of PIP in yeast and the separation and purification of product

Transformed yeast cells is inoculated in 5ml not to be contained in the YPD substratum of agar, cultivate after one day for 30 ℃ and be transferred to the 250mlYPD substratum, 30 ℃ are continued to cultivate two days expression [B9Glu, B10Asp] PIP, expression product is secreted in fermented liquid, and expression amount is measured with Regular Insulin radioimmunity medicine box (Shanghai Vaccine and Serum Institute's product).The centrifugal thalline that goes is used trichloroacetic acid precipitation, and (2.6 * 100cm) separate precipitation, with lmole/L acetic acid wash-out with centrifugal collection back dissolving and by the SephadexG-50 post.Collect [B9Glu, B10Asp] PIP peak, freeze-drying further separates obtaining HPLC pure (Fig. 5 a) and pure (Fig. 4 [B9Glu, B10ASp] PIP a) of electrophoresis with HPLC.

Embodiment 3.[B9Glu, B10ASp] trypsinase of PIP changes peptide and [B9Glu, B10Asp] insulin human's evaluation

With [B9Glu, B10Asp] PIP dissolving what 1 of purifying, 4-butyleneglycol/dimethyl sulfoxide (DMSO)/water (volume ratio 70: 15: 15), the concentration of [B9Glu, B10Asp] PIP is 50mg/ml, adds the excessive 100 times Thr (Bu of mol ^t)-OBu ^t).PH value of solution is transferred to 6.5, add the TPCK-trypsin for [B9Glu, B10Asp] PIP weight 1/5th).Reaction solution adds acetone and makes protein precipitation 25 ℃ of insulations 6 hours.With semipreparative column RP-300 on Beckman HPLC instrument, separate (Fig. 6) wherein peak 1 be unreacted [B9Glu, B10Asp] PIP, peak 2 is for removing B30[B9Glu, B10Asp] insulin human, peak 3 is to change peptide prod B30Thr (Bu ^t) OBu ^t[B9Glu, B10Asp] insulin human (Fig. 4 b).Collect peak 3, go protecting group promptly to get [B9Glu, B10Asp] insulin human (Fig. 4 b, Fig. 5 b) (protein concentration is pressed the 276nm determination of uv absorption) with trifluoroacetic acid.

Get [BgGlu, B10Asp] insulin human's sample of an amount of purifying, in 110 ℃ of hydrolysis 24 hours, the evaporate to dryness hydrolyzed solution was measured amino acid with automatic analyzer for amino acids and is formed with 5.7NHCl.Its amino acid is formed conform to theoretical value (table 1).

Embodiment 4.[B9Glu, B10Asp] insulin human's biologos measures

[B9Glu, the B10Asp] insulin human of purifying and people's placenta cells film insulin binding ability by Feng wait (Feng Youmin etc., Acta Biochimica et Biophysica Sinica, 1982,14:137) method mensuration, the result is as shown in Figure 7.To replace ¹²⁵During I-Regular Insulin 50%, the amount of required unmarked sample is calculated, and [B9Glu, B10Asp] insulin human is 34.4% of a pork insulin. shows 1.[B9Glu; B10Asp] actrapid monotard's amino acid composition analysis amino acid [B9Glu, B10Asp] actrapid monotard Asp 3.896 (4) * Thr 2.881 (3) Ser 2.189 (2) Glu 8.168 (8) Gly 4.000 (4) Ala 0.971 (1) Val 4.088 (4) Ile 1.998 (2) Leu 6.173 (6) Tyr 4.133 (4) Phe 2.877 (3) Lys 0.962 (1) His 0.906 (1) Arg 0.915 (1)

* in the bracket theoretical residue number.

[B9Glu, B10Asp] insulin human's overall activity is measured its overall activity identical with pork insulin (table 2) with sxemiquantitative small white mouse convulsions method.

Small white mouse convulsions reaction vigor in table 2. pork insulin and [B9Glu, B10Asp] insulin human's the body

Measure

Sample concentration (μ g) reaction vigor *

Pork insulin 1.0 10/10

0.5 8/10

0.25 2/10[B9Glu, B10Asp] insulin human 1.0 10/10

0.5 7/10

0.25 2/10* produces reaction mouse number/administration mouse number.

Claims

1, a kind of [B9Glu, B10Asp] insulin human is characterized in that the 9th on the B chain and the 10th amino acids of Regular Insulin is acidic amino acid.

2, by the described insulin human of claim 1, it is characterized in that the 9th of insulin B chain is L-glutamic acid, the 10th is Aspartic Acid.

3, claim 1 or 2 described insulin humans' production method, it is characterized in that it being to make with protein engineering method, its step comprises: the pork insulin precursor-gene is suddenlyd change, mutant primer is 5 '-CTTGTGCGGTGAAGACTTGGTTGAGGC-3 ', and this muton is transformed into yeast, forming preservation registration number is the yeast strain of CCTCC M95042, obtains described insulin human by expressing then.

4, express the yeast strain that obtains claim 1 or 2 described insulin humans, it is characterized in that it being that preservation registration number is the yeast strain Saccharomyces cerevisiae YS92/pLL[B9E of the expressive plasmid that contains pork insulin precursor mutator gene of CCTCC M95042, B10D] PIP (XV-700-6B).