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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 537-542 |
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doi:10.1111/j.1745-7270.2006.00198.x |
B22 Glu Des-B30 Insulin: A
Novel Monomeric Insulin
Hai-Juan DU1,3,
Jia-Hao SHI1, Da-Fu CUI1,
and You-Shang ZHANG1,2*
1
Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological
Sciences, Chinese Academy of Sciences, Shanghai 200031, China;
2 Institute of Protein Research, Tongji
University, Shanghai 200092, China;
3
Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China
Received: March 10,
2006�������
Accepted: May 16,
2006
*Corresponding
author: Tel, 86-21-54921237; E-mail, yszhang @sibs.ac.cn
Abstract������� Studies on monomeric insulin with reduced self-association
are important in the development of insulin pharmaceutical preparations with
rapid hypoglycemic action on patients with diabetes. Here we report a novel
monomeric insulin, B22 Glu des-B30 insulin, prepared from a single chain
insulin precursor with B22 Arg mutated to Glu, which was expressed in Pichia
pastoris and converted to B22 Glu des-B30 insulin by tryptic digestion. It
still retains 50% of the in vivo biological activity of porcine insulin
and does not form a dimer even at a concentration of 10 mg/ml, showing that B22
Glu plays a key role in reducing the self-association of the insulin molecule
without greatly reducing its biological activity. This novel monomeric insulin
might have potential applications in the clinic.
Key words������� monomeric insulin; B22 Glu des-B30 insulin;
self-association
Insulin is a globular protein composed of two poly�peptide chains and three disulfide bonds [1]. In the pancreatic islet� cells, insulin is stored as Zn-insulin hexamers. In the bloodstream, insulin is highly diluted and dissociates rapidly� into monomers, which bind to the insulin receptor [2]. In insulin preparations of therapeutic concentration, insulin exists as dimers and hexamers [3]. After insulin injection, there is a delay in insulin absorption with an initial lag phase followed by dissociation of insulin oligomers with a gradual increase in absorption rate [4]. Therefore, monomeric insulin� with reduced self-association has the benefit of rapid action in treating patients with diabetes.
Porcupine insulin and guinea pig insulin have unusual low potency and low self-association. It was suggested that the residue B22 Asp might be responsible for these unusual properties [5,6]. However, when B22 Arg of porcine insulin was replaced by Asp, the product still retained 50% of the in vivo insulin activity as shown by our previous studies [7,8]. Hence, it is worth studying whether the replacement of B22 Arg by Glu will affect its self-association behavior. It is well known that the removal of B30 residue by tryptic digestion has no effect on the potency, conformation or self-association of insulin. Here, we report the preparation and characterization of B22 Glu des-B30 insulin. It was found that B22 Glu des-B30 insulin possessed 50% of the in vivo biological activity of porcine insulin and did not self-associate under our experimental conditions of neutral pH and high concentration.
Materials and Methods
Construction of Pichia
pastoris expressing B22 Glu insulin precursor
The B22 Arg codon AGA of the single chain insulin precursor gene in plasmid pPIC9K/PIP, constructed in our laboratory for secretory expression of single chain insulin precursor [9], was mutated to Glu codon GAA by PCR method. Two overlapped double-stranded DNA fragments containing the mutated B22 codon were made separately using pPIC9K/PIP as the template and the following pairs of nucleotides as the primers: TTCGAAGGATCCATGAGA/AGAAACCTTCTTCACCGCA; and GGTTTGCGGTGAAGAAGGTT/GGCAAATGGCATTCTGACATC. Using these DNA fragments as the template and TTCGAAGGATCCATGAGA/GGCAAATGGCATTCTGACATC as the primers, the full length of DNA containing the mutated gene was made, which was cleaved by BamHI/EcoRI digestion and cloned into pPIC9K to obtain plasmid pPIC9K/EPIP. The mutated gene was confirmed by DNA sequencing. The plasmid pPIC9K/EPIP was linearized by BglII digestion and cloned into P. pastoris GS115 by electroporation. Transformant with a high copy number of mutated gene integrated into the chromosome was selected [10]. The expression of B22 Glu insulin precursor was detected by pH 8.3 native PAGE.
Secretory expression and
purification of B22 Glu insulin precursor
A strain of phenotype GS115 (His+Mut+) was cultured in a 16 liter fermentor as described by Wang et al. [9]. After centrifugation (6430 g for 20 min) of the culture, the supernatant was applied to an XAD-7 column and washed with 5% acetic acid to remove impurities. The expression product was eluted from the column with 45% ethanol/5% acetic acid. The eluent was concentrated in a rotating evaporator and freeze-dried. The lyophilized powder� was dissolved in 1 M acetic acid and the expressed precursor was precipitated with trichloroacetic acid, followed� by gel filtration using a Sephadex G-25 column. The product was further purified with HPLC using a C18 column (4.6 mm250 mm; Beckman) under the following� conditions: solvent A (0.1% trifluoroacetic acid); solvent B (70% acetonitrile containing 0.08% trifluoroacetic acid); elution gradient 35%-50% of solvent B in 30 min; flow rate, 2 ml/min; and wavelength, 280 nm.
Conversion of the precursor
into B22 Glu des-B30 insulin
The purified precursor at a concentration of 10 mg/ml was converted to B22 Glu des-B30 by tryptic digestion in 0.1 M ammonium bicarbonate at 4 �C overnight, the ratio of substrate to enzyme being 200:1 (W/W). The product purified by HPLC was homogeneous in native pH 8.3 PAGE.
Bioassay of B22 Glu des-B30
insulin
The in vivo biological activity was measured by mouse
convulsion assay and rabbit blood glucose lowering assay in the Chinese
Pharmacopoeia 1985.
Circular dichroism analysis
Samples (3.5�10-5 M) in pH 7.4 Gomori buffer were measured in a Jasco-715 spectropolarimeter at room temperature. The far-UV spectra were scanned from 250 to 190 nm in a cell of 0.1 cm path length and the near-UV spectra scanned from 300 to 245 nm in a cell of 1.0 cm path length.
Determination of
self-association by size exclusion chromatography
The experiment was carried out as reported previously [11] under the following conditions: Superdex 75 column (HR 10/30); 0.1 M ammonium bicarbonate buffer, pH 7.8 or phosphate-buffered saline, pH 7.4; flow rate 0.4 ml/min; room temperature; and detection at 230 nm. All the samples were zinc free. For molecular weight estimation, the distribution coefficient was calculated according to the following Equation 1:
Eq. 1
where Vr is the retention volume, V0 the void volume, and Vc the total bed volume. Molecular weight was estimated from KD vs. logarithmic molecular weight plot.
Results
Conversion of the purified
precursor into B22 Glu des-B30 insulin
The purified precursor was cleaved by trypsin and purified by HPLC. The final product was a single band in native pH 8.3 PAGE (Fig. 1). The molecular mass (5677.8) determined with an ABI API2000 Q-trap mass spectroscope was consistent with the theoretical value.
Biological activity
The in vivo biological activity of B22 Glu des-B30 insulin determined by mouse convulsion assay (Table 1) and rabbit blood glucose lowering assay (Fig. 2) was approximately 50% of porcine insulin activity.
Circular dichroism analysis
The far-UV spectrum of B22 Glu des-B30 insulin showed distinct differences from that of des-B30 insulin (Fig. 3). The negative ellipticity at 222 nm decreased and almost disappeared, and that at 208 nm increased. The ellipticity at 200 nm also reduced and there was a blue-shift between 210 and 200 nm. These results indicate that B22 Glu des-B30 insulin has a lower a-helix content and looser conformation, consistent with a previous report [12] that monomeric insulin had less negative ellipticity at 222 nm and more negative ellipticity at 208 nm. The near-UV spectrum of B22 Glu des-B30 insulin also changed remarkably. There was no obvious negative peak and the negative ellipticity at 275 nm reduced. It was known that the dissociation of insulin oligomer into monomer was reflected by a weakening of the negative peak at 275 nm arising from B16 and B26 tyrosines at the monomer-monomer interface [13-16].
Self-association studied by
size exclusion chromato�graphy
Fig. 4 and Table 2 show the size exclusion chromato�graphy profiles of B22 Glu des-B30 insulin and porcine insulin. The apparent molecular weight of B22 Glu des-B30 insulin estimated by the KD vs. molecular weight plot was found to be 6025, corresponding to the theoretical value 5768 of the monomer (Fig. 5). In contrast to porcine� insulin, the peak was symmetrical and the retention volume� was concentration-independent.
Discussion
Here we report the expression and characterization of monomeric B22 Glu des-B30 insulin. It has 50% of the in vivo biological activity of porcine insulin, similar to that of B22 Asp insulin reported earlier in our laboratory [7,8]. The mutation of B22 Arg with positive charge to Asp with negative charge has such remarkable effect that B22 Glu des-B30 insulin exists as a monomer in solution of neutral pH and high concentration. We would expect B22 Asp insulin is also monomeric, although this was not studied in our earlier work. The mutation of Arg to Glu has the advantage that the peptide bond after B22 will not be cleaved by trypsin. Therefore, the expressed single chain precursor can be converted easily to B22 Glu des-B30 insulin by tryptic digestion, which is much more efficient than tryptic transpeptidation to obtain wild-type insulin. In addition, the deletion of B30 by tryptic digestion was known to have little effect on insulin conformation and activity. X-ray structural analysis [17,18] showed that B22 Arg was in the B20-B23 b-turn and the B24-B26 intermolecular anti-parallel b-sheet was important for dimer formation. The conformation of the b-turn and b-sheet might be destroyed by the mutation of B22 Arg to Glu as shown by circular dichroism studies. Using the methods of Yang et al. [19] and Chang et al. [20], the a-helix content was found to decrease by approximately 10%, whereas the b-sheet content increased by approximately 14% compared with des-B30 insulin.
In conclusion, the present study demonstrates that B22 Glu des-B30 insulin is a novel monomeric insulin with 50% of the in vivo biological activity of porcine insulin. It might have potential applications in the clinic.
Acknowledgement
We are grateful to Ms. Xiao-Xia SHAO of Tongji University (Shanghai, China) for determining the molecular weight by mass spectroscopy.
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