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|Title:||Optimization of Conditions for the Folding and Bioprocessing of Different Derivatives of Human Insulin|
|Abstract:||During the present work we have investigated the regiospecificity of acylation of human insulin using reagents of two different chain lengths and have developed chemogenetic approaches to the preparation of acylated proinsulin derivatives. These were then converted into insulin modified at the ɛ-amino group of Lys29B. For the acylation of human insulin (Sigma) and proinsulin derivative, esters of N-hydroxysuccinimde (Nsuccinimidyl acetate and N-succinimidyl laureate) were used The reaction of N-succinimidyl acetate with insulin was studied using different ratios of the reagent and protein and at various pH values. The MALDI-TOF analysis of the crude reaction mixture showed the formation of mono and di acetyl insulin in about equal amounts, while tri acetyl insulin was present as a minor product. Thiolytic cleavage of these derivatives led to the separation of the two chains and showed that the mono acetyl insulin contained the acetyl moiety only in the B-chain, which was located at the ɛ-amino group of K29B while the di-acetyl insulin following separation of the two chains was acetylated in both the chains. Next, the above protocol was extended to acylation using reagent with a C12 chain length, N-succinimidyl laureate. The MALDI-TOF spectrum of a typical experiment showed the presence of mono as well as di dodecanoyl species, with the predominance of the former. The thiolytic cleavage of mono dodecanoyl insulin showed that the modification was on the B-chain and its tryptic digest analysis, following thiolysis, established that the residue modified by the reagent was present in the octa peptide fragment constituting residues G23B to T30B in the B-chain of insulin. Since the only amino group in this part of insulin is the amino group of K29B this must have been acetylated. From the profile of acylation, found above, it was concluded the ε-amino group Lys29B is the least hindered and accessible to C2 as well as C12 reagents, then is the amino group of Gly1A which is accessible to C2 but not the C12 reagent, finally that of Phe1B which is most hindered and accessible to neither. Native human proinsulin contains three sites for N-acylation; its N-terminal amino group, Lys64 in the C-peptide region and Lys29 destined to become Lys29B xxvii in the derived insulin Our projected objective required the availability of proinsulin derivatives which contained minimum number of N-acylation sites, necessitating the mutation of Lys29by a residue lacking an amino group, yet maintaining the characteristics of the dibasic residues, Arg65- Lys64, required for the removal of the Cpeptide. Furthermore, the N-terminal Met which will be the integral part of any genetically produced protein in E. coli is also likely to be modified during the acylation of proinsulin at Lys29, and should be present in a sequence that is removed during the processing of proinsulin by a single-pot reaction involving trypsin cum carboxypeptidase B, generating the N-terminal Phe of the B-chain of insulin. Initially, we produced proinsulin mutants, in which Lys64 was changed to Arg64 and the Nterminal contained five different linkers which should be removable by trypsin during the excision of the C-peptide These are designated as MR-(R64) hpi, MRR-(R64) hpi, MTRR-(R64) hpi, MFTRR-(R64) hpi and MHHR-(R64) hpi. E. coli BL 21 codon plus, harboring pET21a derivatives encoding the proteins, gave good expression of the desired proteins which were found in inclusion bodies. The proteins were solubilized in 8 M urea and refolded using 1: 10 molar ratio of cysteine: cysteine. The overall yield of the correctly folded proteins, based on the proinsulin polypeptide content was 30%. These mutants [MR-(R64) hpi, MRR-(R64) hpi, MTRR- (R64) hpi, MFTRR-(R64) hpi and MHHR-(R64) hpi] were purified to homogeneity by sepharose Q chromatography followed by RP-HPLC and gave the predicted masses on analysis by MALDI-TOF. These all were then converted into insulin and again analyzed be MALD-TOF. One problem with all of the mutants [MR-(R64) hpi, MRR- (R64) hpi, MTRR-(R64) hpi, MFTRR-(R64) hpi and MHHR-(R64) hpi] was that during the excision of the C-peptide the cleavage at the C/A junction occurred not only at, the desired, R65-G66 bond but also between R64-R65 thus yielding insulin as well as another species in which insulin contained an Arg residue at its A-chain. In order to circumvent the unwanted cleavage between R64-R65, we searched for a mutant which will contain a single tryptic site at the C/A junction, yet maintain the intrinsic properties of the dibasic amino acids at this site to give the folding profile expected from the native sequence. The choice fell on glutamine at position 64, and K64-Q64 mutants containing the four linkers [MRR-(Q64) hpi, MTRR-(Q64) hpi, MFTRR- (Q64) hpi, MHHR-(Q64) hpi] were engineered which following characterization of the xxviii DNA sequences were expressed, the proteins refolded and purified as above. In general, the protein profile of these K64-Q64 mutants was similar to that noted for the K64-R64 series. With respect to processing by trypsin cum carboxypeptidase B, the linker from MRR-(Q64) hpi was removed most smoothly With the encouraging results above, MRR-(Q64) hpi was selected for further studies and treated with acylating agents of two chain lengths used above In the case of modification with N-succinimidyl acetate, mono and di acetylated derivatives of MRR- (Q64) hpi were produced in the ratio of 1:1. These when treated with trypsin and carboxypeptidase B, singly or as a mixture, led to a smooth processing of the linker as well the C-peptide producing mono acetyl insulin. It is gratifying that, as hoped for, the linker whether contained a free or an acylated amino group at N-terminal was removed with equal facility. Reaction of N-succinimidyl laureate with MRR-(Q64) hpi, predominantly led to the formation of mono dodecanoyl insulin, containing modification at the ε-amino group of K29; di derivative with modifications at the ε-amino group of Lys29 and N-terminal methionine was produced as a minor product. Treatment of the mono derivative or of the mixture containing the di derivative with trypsin cum carboxypeptidase b gave dodecanoyldes-30 insulin. The biological activity of the modified and unmodified insulins, prepared in the present study, was determined and it was found that these were as active as reference derivatives.|
|Gov't Doc #:||16492|
|Appears in Collections:||PhD Thesis of All Public / Private Sector Universities / DAIs.|
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