Expression, Purification And Pharmacologic Properties Of Pro-rhglp-1, A Prodrug Of Recombinant Human Glucagon-like Peptide-1

BackgroundDM is a serious threat to human health associated with increased morbidity and mortality, and is occurring at younger and younger ages. About 95% of patients are suffering from T2DM, which is in poor glycemic control. Sulfonylurea, metformin and other oral hypoglycemic medications were used as the first-line therapy in the patients with T2DM. However, the most common and dangerous adverse effect of these drugs is persistent hypoglycemia, and long-term treatment may be lead toβ-cell failure caused by excessive secretion of insulin. Moreover, the traditional treatment for T2DM only focus on blood glucose reduction, which lead to most patients are unable to reverse the declining glycemic control,β-cell mass and function. Therefore, it is very important to research and develop more effective new drug for the treatment of diabetes associated a variety of symptoms and complications. ObjectiveGLP-1, an important gut-derived incretin hormone, plays numerous important roles in regulating blood glucose homeostasis. The main actions of GLP-1 are to stimulate insulin secretion and inhibit glucagon secretion, thereby decreasing high glucose. GLP-1 also appears to stimulateβ-cell proliferation, improve insulin resistance and inhibit gastric emptying and food intake. In addition, GLP-1 has both neuro- and cardioprotective effects. It has been proposed as a promising drug candidate for diabetes treatment. However, what so far is hampering the therapeutic potential of GLP-1 is the very rapid degradation of the native peptide in plasma by DPP IV. Therefore, current development focused to prolong the duration of action of GLP-1. Based on this, we designed Pro-rhGLP-1 by polymeric prodrug strategy and produced it by DNA recombinant technology. Pro-rhGLP-1 is a prodrug of recombinant human GLP-1 consists of repeated bioactive GLP-1 linked by linker peptide. The purpose of the present study was to establish a method for high-level expression and purification of Pro-rhGLP-1, and to characterize the pharmacologic properties of Pro-rhGLP-1, determing its long-lasting effects on glycemic control.Methods1. Expression, purification and bioactivity of Pro-rhGLP-1 tag fusion protein: The synthetic Pro-rhGLP-1 gene was subcloned into prokaryotic expressing vector pET32a(+) fused with Trx·Tag and His·Tag to construct a recombinant plasmid pET32a(+)-Pro-rhGLP-1, then transformed into E.coli BL21(DE3). The constructed E.coli BL21(DE3)/pET32a(+)-Pro-rhGLP-1 was induced by IPTG. The target protein expressions was identified by 12% SDS-PAGE analysis. The inclusion body was washed followed the sequence of 2-8 M urea in 0.02 M PBS. Then the protein in urea solution was renaturated by dilution refolding. The refolded protein was purified by Ni-NTA affinity chromatography, then the fusion tags were removed by enterokinase. The purity of Pro-rhGLP-1 was assessed by SDS-PAGE and HPLC analysis. The specificity was identified by western blot. The biological activity of Pro-rhGLP-1 was examined by OGTT in C57BL/6J mice.2. Expression, purification and bioactivity of Pro-rhGLP-1 tag-free recombinant protein: PCR technology was used to amplify Pro-rhGLP-1 DNA, and the product was ligated into pET41a(+) removed tags. The fragments were made flush-ended by the Klenow fragment, ligated, and used to construct a recombinant plasmid pET41a(+)-Pro-rhGLP-1(Tag-free), then transformed into E.coli BL21(DE3). The constructed E.coli BL21(DE3)/pET41a(+)-Pro-rhGLP-1 (Tag-free) was induced by 0.01 mM IPTG at 37℃for 4 h. The inclusion body was washed followed the sequence of Triton X-100, 2-8 M urea in 20 mM Tris-HCl, and then dissolved by 8 M urea. The protein in 8 M urea was purified by Q-Sepharose FF anion exchange and Superdex 75 gel filtration chromatography. The purified protein was renaturated by dilution refolding and removed endotoxin. The purity of Pro-rhGLP-1 was assessed by HPLC analysis and the specificity was identified by western blot. The biological activity of Pro-rhGLP-1 was examined by OGTT in C57BL/6J mice. The structure of Pro-rhGLP-1 was confirmed by analysis of amino acid composition, N-terminal and C-terminal amino acid sequence, peptide map and MALDI-TOF-MS. The stability of the engineering strain was analyzed after the 50th generation.3. The pharmacologic properties of Pro-rhGLP-1: The in vitro release kinetics of GLP-1 from Pro-rhGLP-1 was analyzed by western blot. Plasma GLP-1 levels following a single administration of Pro-rhGLP-1 were determined by ELISA. The in vitro effects of Pro-rhGLP-1 were evaluated using isolated pancreatic islets. The acute effects on glycemic control and food intake were investigated in C57BL/6J mice s.c. administered with Pro-rhGLP-1. The chronic effects of Pro-rhGLP-1 on glycemic control were further assessed in C57BL/6J and db/db mice treated twice daily for 6 weeks.Results1. Expression, purification and bioactivity of Pro-rhGLP-1 tag fusion protein: The construction of recombinant expression plasmid pET32a(+)-Pro-rhGLP-1 fused with Trx·Tag and His·Tag was transformed into E.coli BL21(DE3). Pro-rhGLP-1 tag fusion protein was induced by 0.01,0.1 and 1.0 mM IPTG and its molecular weight was about 35 KD analyzed by SDS-PAGE. The expression of Pro-rhGLP-1 was approximately 50% of the total bacterial proteins and was found as insoluble aggregates in inclusion body. A small amount of inclusion bodies could be dissolved in 0.02 M PBS without urea, and about 80% of inclusion bodies were dissolved by 8 M urea. The purity of Pro-rhGLP-1 was more than 95.43% by Ni-NTA affinity chromatography. The analysis of in vivo activity indicated that Pro-rhGLP-1 significantly decreased blood glucose and increased insulin secretion in dose-dependent manner, and the effects were more potent than that produced by GLP-1.2. Expression, purification and bioactivity of Pro-rhGLP-1 tag-free recombinant protein: The construction of recombinant expression plasmid pET41a(+)-Pro-rhGLP-1(Tag-free) was transformed into E.coli BL21(DE3). Pro-rhGLP-1 tag fusion protein was induced by 0.01,0.1 and 1.0 mM IPTG and its molecular weight was about 19 KD analyzed by SDS-PAGE. The expression of Pro-rhGLP-1 was approximately 60% of the total bacterial proteins and was found as insoluble aggregates in inclusion body. The soluble expression of target protein was not obtained by reducing induction temperature and IPTG concentration. The inclusion bodies was in poor solubilization using a variety of solutions at different pH values. More than 90% of inclusion bodies were dissolved by 8 M urea. The target protein of Pro-rhGLP-1 was purified by anion exchange and gel filtration chromatograph. The purity was 97.55% by HPLC. The biological activity of Pro-rhGLP-1 was determined after dilution refolding. It showed that Pro-rhGLP-1 significantly decreased blood glucose and increased insulin secretion in dose-dependent manner, and the effects were more potent than that produced by GLP-1. The results of structure comfirmation were consistent with expectations. The stable and specific expression of Pro-rhGLP-1 was detected after the 50th generation of engineering strain.3. The pharmacologic properties of Pro-rhGLP-1: (1) GLP-1 was initially released from Pro-rhGLP-1 after 15 min incubation and gradually increased by continuous degradation following 12 h. After 12 h incubation, Pro-rhGLP-1 was degraded with approximately 80% giving rise to GLP-1. (2) The increment of plasma GLP-1 could still be detected 12 h after a single s.c. injection of Pro-rhGLP-1 in C57BL/6J mice. (3) Pro-rhGLP-1 did not exhibit the insulinotropic action in isolated pancreatic islets without plasma, but GLP-1 caused a significant concentration-dependent increase in glucose-stimulated insulin secretion. (4) The glucose-lowering actions of Pro-rhGLP-1 (3 nmol/kg) remained no less than 12 hours after a single injection to diabetic db/db mice. Pro-rhGLP-1 administered for 6 weeks significantly reduced HbA1c in diabetic mice, and remained so for a further 6 weeks. (5) Pro-rhGLP-1 dose-dependently increased insulin secretion. IGI was increased 8.2-fold by Pro-rhGLP-1. (6) Pro-rhGLP-1(3nmol/kg) inhibited 24-h food intake by 51±11% in C57BL/6J mice and caused a durable inhibition of food intake in db/db mice by up to 32% following 6-week administration. (7) Pro-rhGLP-1 significantly reduced body weight, body fat, and plasma leptin, TG and NEFA levels. (8) HOMA-IR was significantly decreased while QUICKI was increased in db/db mice. (9) Pro-rhGLP-1 significantly increased both the percentage of islet areas and number of islets, and stimulatedβ-cell proliferation in db/db mice.Conclusions1. We first designed a prodrug of GLP-1 by prodrug strategy, termed as Pro-rhGLP-1. A prokaryotic expression system of Pro-rhGLP-1 tag-free recombinant protein with high efficiency was successfully established and the target protein was obtained in a purity of > 95%. It provides an easy way to produce a novel anti-diabetic drug.2. Pro-rhGLP-1 was inactive in vitro. But when entering body, this prodrug was slowly digested and the active GLP-1 was gradully released for at least 12 h.3. Pro-rhGLP-1 dose-dependently increased insulin secretion and decreased glucose, but did not exhibit the insulinotropic action in isolated pancreatic islets without plasma. Pro-rhGLP-1 could be remained in a"silent"state below normal blood glucose level. The glucose-lowering actions of Pro-rhGLP-1 remained no less than 12 hours after a single injection. The chronic treatment with Pro-rhGLP-1 improved glucose tolerance and insulin sensitivity, increasedβ-cell mass and proliferation, and inhibited food intake and body weight gain.4. Pro-rhGLP-1 has long-lasting and potent effects on glycemic control, promotion of islet function, and improvement of insulin sensitivity and glucose and lipid metabolism disorders. It offers significant advantages over currently available agent for the treatment of T2DM and obesity.