Monomeric
B27 Lys Destripeptide Insulin: Semisynthesis, Characterization and Biological
Activity

DING
Jin-Guo, CUI Da-Fu, ZHANG You-Shang*

( Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences,

 the Chinese Academy of Sciences, Shanghai 200031, China )

Abstract  In
this paper, we report the semisynthesis of B27 Lys destripeptide insulin (B27
Lys DTrI), i.e. destetrapeptide insulin with an additional Lys residue at the
C-terminus of B-chain. B27 Lys DTrI is also monomeric as shown by gel
filtration. Its in vivo biological activity is 80% in comparison with
that of native insulin. The addition of a Lys residue at the C-terminus of
B-chain makes it possible to obtain monomeric B27 Lys DTrI from a precursor
expressed in Saccharomyces cerevesiae by tryptic hydrolysis instead of the less
efficient tryptic transpeptidation.

Key
Words B27 Lys
destripeptide insulin; biological activity; monomeric insulin

The incidence of diabetes is ever
increasing in China and in other countries. Native insulin and novel insulin
with improved therapeutic properties prepared by protein engineering have been
used effectively to treat diabetes. Native insulin preparations used in the
clinic exist in the form of dimer and hexamer while the functional unit of
insulin is monomer. The absorption of native insulin oligomers to the blood
stream and their conversion into monomers are time consuming. Therefore,
monomeric insulin, i.e. active insulin analogue in the form of monomer has the
advantage of fast acting in comparison with native insulin.

Among the monomeric insulins,
deshexapetide insulin (DHI, human insulin with B25-30 removed) and
despentapeptide insulin (DPI, human insulin with B26-30 removed) were found to
be active^[1-3]. Later, Brems et al.^[4] reported
that removal of B28-30 or B27-30 from insulin resulted in much less
self-association.

We have prepared destetrapeptide insulin
(DTI, human insulin with B27-30 removed) from a precursor expressed in
Saccharomyces cerevisiae and it was shown that DTI was monomeric^[5].
Here, we report the preparation of another monomeric insulin, B27 Lys DTrI,
i.e. insulin with B28-30 removed and B27 Thr replaced by Lys. With a Lys as the
C-terminus of B-chain, it can be obtained from its precursor expressed in yeast
through tryptic hydrolysis instead of tryptic transpeptidation.

1 Materials and Methods

1.1 Materials

Crystalline porcine insulin was purchased from Nova.
TPCK-trypsin, N-hydroxysuccinimide (N-HOSu), dimethylsulfone
(DMSO) and trifluoroacetic acid (TFA) were products from Sigma. 1, 4
butanediol, Boc-amino acids and dicyclohex cylcarbodiimide (DCCI) were products
from Japan Peptide Institute. [28 Lys, B29 Pro] -insulin was a gift from Tang
Yuehua in the Institute of Biochemistry and Cell Biology, Shanghai Institutes
for Biological Sciences. Sephadex G50 fine, DEAE-Sephadex A25 and Superdex 75
HR10/30 were from Pharmacia. HPLC instrument was Hewlett Packard Series 1050.
Other reagents were AR grade.

1.2 Thin layer
chromatography

It was performed on silica gel plate. The developing
solution was a mixture of 3 volumes of solution A (pyridine:acetic acid:water =
4:1:1.5) and 7 volumes of solution B (ethyl acetate_isopropanol=10:4). The
chromatogram was stained by chlorine-KI-starch or ninhydrin.

1.3
Polyacrylamide gel electrophoresis

It was performed according to the method of Gabriel^[6],
pH 8.3, gel concentration 15%, stained with Coomassie brilliant blue.

1.4 HPLC

The mobile phase Solution A was distilled water with
0.1% TFA and solution B was 70% acetonitrile with 0.1% TFA. In the analysis of
Gly-Phe-Phe-Tyr-Lys(Boc)OBut, C8 reverse phase column (4.6 mm×250 mm) was used,
flow rate 1 ml/min, wavelength 280 nm, initial mobile phase solution A and
elution gradient 0%-100% solution B in (0-50 min). In the isolation of B27 Lys
DTrI, C8 reverse phase column (10 mm×250 mm) was used, flow rate 2 ml/min,
wavelength 280 nm, initial mobile phase solution a and elution gradient 10%-60%
solution B (10-40 min). In the analysis of B27 Lys DTrI, C8 reverse phase
column (4.6 mm×250 mm) was used, flow rate 1 ml/min, wavelength 280 nm, initial
mobile phase solution A and elution gradient 30%-70% solution B (10-40 min).

1.5
Determination of protein concentration

The protein concentration was determined by
ultraviolet absorption at 280 nm. A280 for insulin is 1.01 at a concentration
of 1 g/L and a path length of 1 cm, while those for DPI, ［B28
Lys, B29 Pro］-Insulin and B27 Lys DTrI were 0.88, 1.01
and 1.07 respectively.

1.6 Bioassay

The in vivo biological activity was determined
by mouse convulsion assay in Chinese Pharmacopoeia 1985 and by mouse blood glucose
assay in Chinese Pharmacopoeia 2000. The receptor binding assay was determined
as described^[7].

1.7
Determination of self association

It was determined by size exclusion chromatography^[4,5]
using Superdex 75 (HR 10/30) column, eluted by pH 7.4 phosphate buffered
saline, flow rate 0.4 ml/min, sample volume 0.1 ml, monitered at 230 nm. The
molecular homogeneity was measured by symmetry factor Fs. Fs = W0.05h/2A, where W0.05h is the band width at 0.05 peak height, A
is the width of the first half peak at 0.05 peak height. The change of average
molecular weight was measured by plotting the distribution coefficient K_av
vs protein concentration. K_av = (V_r/V_o)/(V_c/V_o), where V_r is the retention volume, V_o the void volume and V_c the total bed volume.

2 Results

2.1 Synthesis
of Gly-Phe-Phe-Tyr-Lys(Boc)-Obut

Cbz-Gly-Phe-Phe-Tyr-OC₂H₅ (m.p.
148–151 ℃) was obtained from Tyr-OC₂H₅, Boc-Phe and
Cbz-Glly with the aid of DCCI and HOSu. Cbz-Gly-Phe-Phe-Tyr-Lys(Boc)-OBut (m.p.
160–162 ℃) was obtained from Cbz-Gly-Phe-Phe-Tyr-OC2H5 and Lys(Boc)-OBut using
azide method. The product was hydrogenated for 6 h to remove the Cbz group. The
reaction mixture was filtered, evaporated, dried under vacuum and washed
thoroughly with ethyl acetate. The product was homogeneous in thin layer
chromatography and C8 column HPLC and ninhydrin positive. Its amino acid
composition was consistent with the theoretical value: Gly 1.2, Tyr 1.0, Phe
2.2 and Lys 1.

2.2
Preparation of B27 Lys DtrI

Desoctapeptide insulin (DOI, human insulin with B23-30
removed) was prepared from zinc free porcine insulin by TPCK-trypsin digestion
and purified by DEAE-Sephadex A-5 chromatography as described^[8].
The tryptic semi-synthesis of B27 Lys DTrI was performed in
1,4-butanediol-DMSO-water system as described^{[2, 9]}. 0.23 g (0.172
mmol) Gly-Phe-Phe-Tyr-Lys(Boc)-OBut was dissolved in 0.26 ml DMSO by warming to
50 ℃. The solution was incubated in 37 ℃ water bath and 86 mg (0.0172 mmol) DOI
was slowly added into the solution. 1.82 ml 1, 4 butanediol and 0.52 ml water
were added to the solution and the pH was adjusted to 6.5 with N-methylmorpholine.
7.8 mg TPCK trypsin was added and the reaction mixture was incubated at 30 ℃.
After 2 h and 4 h, TPCK-trypsin was added, each time 4.5 mg. After 20 h, 1.1 ml
glacial acetic acid was added and the reaction was ended by adjusting pH to 3
with 1 mol/L HCl. The reaction mixture was purified by Sephadex G50 fine column
chromatography with 30% acetic acid as eluent. 76 mg crude product of B27 Lys
DTrI(Boc)-OBut was obtained (80% yield). The crude product was purified by C8
HPLC, then washed with cold acetone and dried. The dried product was dissolved
in anhydrous TFA to a concentration of 3 g/L to remove the blocking group of
Boc and OBut. After standing for 1 h, the TFA was evacuated and the residue was
washed thoroughly with dichloromethane. The product was purified by C8 HPLC to
obtain B27 Lys DTrI.

2.3
Characterization of B27 Lys DtrI

The purified B27 Lys DTrI was homogeneous in pH 8.3 PAGE
(Fig.1) and C18 HPLC. The molecular weight determined by electrospray mass
spectroscopy was 5509 (Fig.2), consistent with the theoretical value 5508.3.
Sequences of 14 N-terminal residues and 2 C-terminal residues in both A and B
chain of B27 Lys DTrI were determined to be correct.

2.4 Biological
activities

The in vivo biological activity of B27 Lys
DTrI determined by mouse convulsion assay was 21 u/mg with FL% (confidence
limit rate) of 23.5%, that determined by mouse blood sugar lowering method was
23 u/mg with FL% of 11.9%. The activity of insulin standard was 27 u/mg.
Therefore, in comparison with native insulin, the in vivo biological
activity of B27 Lys DTrI was 80%.

Fig.1 Polyacrylamide gel electrophoresis at pH 8.3

1, DOI; 2,
reaction mixture of enzymatic semisynthesis; 3, crude B27 Lys DTrI; 4, purified
B27 Lys DTrI; 5, insulin.

Fig.2 Electrospray mass-spectrometric analysis of B27
Lys DtrI

A Finnigan
MATLCQ ESI-MS Instrument was used, spray voltage 4.25 kV, capillary temperature
200 °C.

The receptor binding activity of B27 Lys DTrI was
(125±13)% as shown in Fig.3.

Fig.3 Displacement of labeled insulin from insulin
receptor on human placental membrane by insulin (dot) and B27Lys-DTrI
(triangle)

2.5 Monomeric
behaviour

The monomeric behaviour of B27 Lys DTrI was
determined by size exclusion chromatography. The retention time on Superdex 75
column decreased with increasing concentration of zinc free insulin control as
shown in Fig.4(A); while the retention time of B27 Lys DTrI was independent of
protein concentration as shown in Fig.4(B). The Fs value of insulin increased
markedly at concentration above 400 μmol/L ［Fig.4(C)］
and the Kav value of insulin decreased with increasing concentration ［Fig.4(D)］.

Fig.4 Effect of protein concentration on
size-exclusion chromatography

Zn-free porcine
insulin (A) and B27 Lys DTrI (B), the different concentrations of each protein
injected were 40, 80, 200, 400, 600 μmol/L. Effect of protein concentration on
the the peak shape (C) and elution time (D), insulin (dot) , B27 Lys DTrI
(triangle).

The monomeric behaviours of [B28 Lys, B29
Pro]-insulin, DPI and B27 Lys DTrI were compared at the same concentration (80
μmol/L) on Superdex 75 column. The Kav values of B27 Lys DTrI and [B28 Lys, B29
Pro]-insulin were 0.51 and 0.48 respectively.

3 Discussion

Monomeric insulins have been studied extensively^[10,11].
However, up to now only two are commercially available: one is Humalog from Eli
lilly, which is insulin with B28 Pro and B29 Lys inverted; another is Novo Rapid
from Novo Nordisk, which is insulin with B28 Pro replaced by Asp. So
development of monomeric insulin with high activity is very attractive. The
monomeric B27 Lys DTrI we developed is suitable for large scale production from
its precursor expressed in yeast and may have benefits for diabetics.

More than 4600 kg of insulin are used yearly for the
treatment of patients with diabetes mellitus. The incidence of diabetes is
increasing rapidly and it is estimated that by the year 2025 the number of
diabetic patients in the world will be 300 million. The development of more
monomeric insulins is necessary to meet the ever increasing demand.

Acknowledgements We
thank Dr. ZHANG Xin-Tang for helping receptor-binding bioassay.

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Received: October 14, 2002     Accepted:
November 17, 2002

*Corresponding author:
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