Insulin analogs with reduced affinity to insulin receptor and use thereof

1. An insulin analog comprising an A-chain of SEQ ID NO: 55 of the following Formula 1 and a B-chain of SEQ ID NO: 56 of the following Formula 2, wherein the insulin analog shows binding affinity to insulin receptors of about 99% or less and about 75% or greater, with respect to the binding affinity of native insulin to insulin receptors, as evaluated by Scintillation proximity assay (SPA), the binding affinity of native insulin to insulin receptors being 100%:

Formula 1

(SEQ ID NO: 55)

Xaa1-Ile-Val-Glu-Xaa5-Cys-Cys-Thr-Ser-Ile-Cys-

Xaa12-Leu-Xaa14-Gln-Xaa16-Glu-Asn-Xaa19-Cys-Xaa21

Formula 2

(SEQ ID NO: 56)

Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu

-Ala-Leu-Xaa16-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-

Xaa25-Tyr-Xaa27-Xaa28-Lys-Thr,

wherein

(1) in Formula 1, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is histidine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in Formula 2, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(2) in Formula 1, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is lysine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in Formula 2, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline;

(3) in Formula 1, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is alanine, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in Formula 2, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline; or

(4) in Formula 1, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in Formula 2, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

2. The insulin analog of claim 1 , comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 28, 30, 44, and 46.

3. The insulin analog of claim 1 , wherein

in Formula 1, Xaa1 is glycine, Xaa5 is glutamine, Xaa12 is serine, Xaa14 is aspartic acid, Xaa16 is leucine, Xaa19 is tyrosine, and Xaa21 is asparagine; and in Formula 2, Xaa16 is tyrosine, Xaa25 is phenylalanine, Xaa27 is threonine, and Xaa28 is proline.

4. An isolated nucleic acid encoding the insulin analog of claim 1 .

5. A recombinant expression vector comprising the nucleic acid of claim 4 .

6. A transformant comprising the recombinant expression vector of claim 5 .

7. The transformant of claim 6 , wherein the transformant is E. coli.

8. A method of preparing the insulin analog of claim 1 comprising:

a) expressing the insulin analog of claim 1 by culturing a transformant comprising a nucleic acid encoding the insulin analog; and

b) isolating and purifying the expressed insulin analog.

9. The method of preparing the insulin analog of claim 8 , wherein the b) the isolating and purifying comprise:

b-1) obtaining the transformant from the culture in step a) and pulverizing the same;

b-2) recovering the expressed insulin analog from the pulverized cell lysate followed by refolding the same;

b-3) purifying the refolded insulin analog by cation exchange chromatography;

b-4) treating the purified insulin analog with trypsin and carboxypeptidase B; and

b-5) sequentially purifying the treated insulin analog by cation exchange chromatography, and anion exchange chromatography or reversed-phase chromatography.

10. A composition comprising the insulin analog of claim 1 as an active ingredient.

11. A method for treating a subject with diabetes, including administering the insulin analog of claim 1 or a composition containing the same to the subject.