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1 V ol. 25 N o CHEM ICAL JOU RNAL O F CH IN ESE UN IV ERS IT IES [B10Glu, B24-A sp-b25 ],,, (,, ) PCR, B B 10 H is Glu, B 24 B 25 A sp, [B 10Glu, B 242A sp2b 25 ]. pbv 220, DH 5Α,, 20%30%.. B, D EA E R P2H PLC.,.,, 73. 6% 146%. ; ; ; Q 816 A (2004) ( molgl ), molgl,,,, [1 ].., E llil illy L isp ro N ovo N ordisk A spart.., B Α B23B 28. B 24Phe B 26T yr., Zn 2+ B10H is, [2 ]. [B10Glu, B 242A sp2b 25 ],, pbv 220. M et2l ys2p rosinsulin pv K100.. E. colidh 5Α. E cor g B am H g, T aq T 4 DNA P rom ega. (1 420 BA EEgm g) Sigm a, B (100 U gm g). 125 I. Sigm a. Pharm acia.. Centus 480 PCR ( Perk in2e lm er ), AB I PR ISM 377 DNA (AB I ), F reezone 6 ( L abconco ), JY922g ( ), ECP3000 (), M ini2pro T EAN g ( B io2r ad ), A β KTA exp lorer ( Pharm acia ), H ew lett Packkard Series 1100 R P2H PL C (A gilent ), B iflex2g M ALD I2TO F2M S ( B ruker ), CD 26 (JOB IN YVON 2 : : (1936 ),,,,. E2m ail: rulab@pku. edu. cn

2 N o. 6 : [B 10Glu, B 242A sp2b 25 ] SPEX ) PCR, 1 ( 5 2GGAA T TCCA TA T2 GAA GT T T GTCAA TCA GCA CCT T T GT GGT TCT GA GCT GGT GGA 23 ), 2 ( 5 2T TCA CGA CG2 CGTCT T GGGT GT GTA GAAA TCGAAA CCA CGT TCCCCA 23 ) 3 ( 5 2GGTCGA CGGA TC2 CTCA GT T GCA GTA GT TCTCCA 23 ). 1 2 (94, 45 s; 55, 1 m in; 72, 1 m in, 25, m in), 3, 25, m in PCR, E cor g B am H g, pbv 220, pbv 2202hP2 M U T 2,. DH 5Α,., 50 ml 50 ΛggmL LB, h, 500 ml 50 ΛggmL LB, h, h g, 10 m in, [ 3 ] molgl Gly2N aoh, 1 mmolgl GSH, 0. 1 mmolgl GSSG, ph = h , 50 mmolgl T ris2hc l, ph = Sephacryl S2100 (60 cm 26 mm ), 50 mmolgl T ris2hc l, ph = , 10 m ggml 80 mmolgl T ris2hc l(ph = 7. 6) B, 3730 m in,, 70%. D EA E Sepharose, 50 mmolgl T ris2hc l(ph = 8. 0), 0. 5 molgl N ac l 50 mmolgl T ris2hc l(ph = 8. 0) molgl N ac l. R P2H PL C, Zorbax R P C18 (4. 6 mm 150 mm ). A 0. 1% T FA 1% ; B 0. 1% T FA 1%. B 0. 5 molgm in 0%60%,,. PA GE,. M ALD I2TO F mmolgl (ph = 7. 2), 1. 2 m ggml. Superdex75 HR 10g30, 20 mmolgl (ph = 7. 2), 200 ΛL, 0. 4 molgm in, 280 nm, mmolgl (ph = 7. 2), CD 0. 2 m ggml, nm, 1 mm. CD 3. 5 m ggml, nm, 1 cm nm gs, , [ 4 ] PCR, pv K100 [B10Glu, B 242A sp2b 25 ] (1) pbv 220 pbv 2202hPM U T 2.,, (2). 2. 2, 15% SD S2PA GE.,,., 20%, (3). 1061

V ol. 25 2. 3, Sephacryl S2100. 15% SD S2PA GE, 9 000 (3). 2. 4 B, D EA E Sepharose, R P2H PL C (4). 90%.

3SD S-PAGE ana lysis of target prote in W hole cell lysates of DH 5Α containing pbv 2202hP2 MU T2; 5. p roinsulin; 6.

3 V ol , Sephacryl S % SD S2PA GE, (3) B, D EA E Sepharose, R P2H PL C (4). 90%. 4, M ALD I2TO F, 9 753, ( ) 0. 1%,, , M et2l ys2 ( ), 0. 1%, M et2l ys, M et2l ys. 14. F ig. 3SD S-PAGE ana lysis of target prote in W hole cell lysates of DH 5Α containing pbv 2202hP2 MU T2; 5. p roinsulin; 6. w hole cell lysates of DH 5Αcontain2 ing pbv 220; 7. w hole cell lysates of DH 5Α. 2. 5,, (5). (1. 2 m ggml ),., [5 ] CD 6 (A ), nm, Α. CD [6 (B) ]275 nm,,, 1062

4 N o. 6 : [B 10Glu, B 242A sp2b 25 ], [6 ] (R IA ), 73. 6% (7)., 146% (8). F ig. 6Far-CD (A) and near-cd (B) spectra of human in sul in (a) and human in sul in ana logue (b) 2. 8, A 21, B 8, 9, 12, 16, 21, 2329, B B24Phe, B 25Phe B26T yr 3 [7 ]., 3. B 10 Zn 2+,. B10 Glu,. B 24 B 25 A sp,,,,. Α2,. CD,, [8 ].,.,, B [9 ]. B Β2Β2B Α2, A 1, 19, 21, B. B C A N, A 2 A 3, [10 ]., B Β2, A sp,.,. [ 1 ]Zinm an B.. N. Engl. J. M ed. [J ], 1989, 321: [ 2 ]HUA Q in2xin, HU Shi2Q uan, B ruce H. F rank et al.. J. M ol. B iol. [J ], 1996, 264: [ 3 ]CHEN J in2q iu, ZHAN G Hong2Tao, HU M ei2hao et al.. App l. B iochem. B iotech. [J ], 1995, 55:

5 V ol. 25 [ 4 ]Fujita2Yam aguchi Y., Choi S., Sakamoto Y.. J. B iol. Chem. [J ], 1983, 258 (8) : [ 5 ]Chen H., ShiM., Guo Z. Y. et al.. P rotein Engineering[J ], 2000, 13 (11) : [ 6 ]David N. B rem s, L eila A. A lter, M ichael J. Beckage et al.. P rotein Engineering[J ], 1992, 5 (6) : [ 7 ]B lundell T., Dodson G., Hodgkin D. et al.. A dv. P rotein. Chem. [J ], 1972, 26: [ 8 ]D iane L. Bakaysa, Jerry Radziuk, Henrt A. Havel et al.. P rotein Science[J ], 1996, 5: [ 9 ]De M eyts P., V an O bberghen E., Roth J.. N ature[j ], 1978, 273 (15) : [ 10 ]M ichael A. W eiss, W an Zhuli, Zhao M ing et al.. J. M ol. B iol. [J ], 2002, 315: Prepara tion, Iden tif ica tion and Properties of [ B10Glu, B24-A sp-b25 ] Human In sul in Ana logues HUA IW en2h ui, SUN T ing2w an, YU M ei2m in, RU B ing2gen 3 (N ational K ey L aboratory of P rotein E ng ineering and P lant Genetic E ng ineering, Colleg e of L if e S ciem e, P ek ing U niversity, B eij ing , China) AbstractD iabetes m ellitus is a k ind of serious endocrinopathy and insulin is the most effective m edicine for the treatm ent of diabeteṡ T he effective form of insulin w hen used as m edicine is monom eṙ How ever, norm al insulin usually form s polym ers because of its h igh self2association, w h ich m akes it lag in the regula2 tion of glucose level in blood. So it is necessary to develop monom eric insulin analogueṡ R esearch on the structure and function of insulin indicates that the interaction betw een the two insulin molecules of dim er insulin exists m ainly betw een the two Β2sheets of C term inus of B chainṡ T here is a strong hydrophobic interaction betw een arom atic rings of B 24, B 25, B 26 residueṡ T he hydrophobic interaction is crucial for the form ation of insulin dim eṙ B 10 H is residue is very important for the form ation of hexam eṙ Based on these points, m utationsw ere introduced to the interaction surface, expecting to get monom eric insulin w ith a low self2association. A m utant gene w as constructed th rough asymm etric PCR. B 10H is residue w as re2 p laced w ith Glu and A sp w as inserted betw een B24 and B25 residueṡ T he gene w as cloned into pbv 220, w h ich is a heat2inducing exp ression vectoṙ T he gene w as exp ressed in E. Coli DH 5Αw ith a h igh level. P rotein w as purified th rough Sephacyl S2100 after denature and refolding. T he p roinsulin analogue w as di2 gested by tryp sin and carboxypep tidase B, the p roduct of w h ich w as purified th rough D EA E Sepharose and R P2H PL C. P rotein identities w ere confirm ed by M ALD I2TO F m ass spectrom etry. Purified insulin analogue w as obtained. T he self2association of the in sulin analogue w as studied th rough analytical size ex2 clusive ch rom atography w ith Superdex 75 colum n. T he result show ed an low er self2association than hum an in sulin obviously. Confo rm ation changes w ere studied th rough circular dicho ism, results of w h ich also show ed the in sulin analogue had a low self2association. T hese results indicated th is in sulin analogue had a strong monom eric p roperty. B10H is w as rep laced w ith Glu, and hexam er could not form. T he acidic residue A sp w as in serted betw een B 24 and B 25. It could destroy the hydrophobic in teraction betw een moleculeṡ T hese two factors could w eaken the self2association of insulin greatly. T he biological activity of the hum an insulin analogue w as also studied in v itro. T he relative activity of R IA w as 73. 7%. T he relative activity of RBA w as 146%. T he analogue retained h igh activities in v itro. It suggested that [B 10Glu, B 242A sp2b25 ] hum an insulin could be a potential drug for the treatm ent of diabeteṡ KeywordsInsulin analogue; M onom eric insulin; P rocaryotic exp ression; B iological activity (Ed. : H, J, Z) 1064

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