Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli

In our previous studies, the recombinant gonadotropin-releasing hormone (GnRH) peptide was constructed into a T7 RNA polymerase-based expression system. The recombinant gene encoding GnRH3-hinge-MVP, which contained three repeated GnRH units, a fragment of hinge region (225-232/225′-232′), and a T cell epitope of measles virus protein, was cloned into Escherichia coli BL21 harboring pED-GnRH3. The high activity of T7 RNA polymerase could make the expression system very powerful for high-level expression of the recombinant protein. However, during the large-scale production of recombinant protein, the productivity of the fermentation process was directly affected by many factors, such as plasmid stability, protein production, and culture conditions. In this study, we studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, the time of induction by lactose, and the number of successive cultures. The results indicate that the plasmid instability may be caused by a loss of plasmid rather than structural change. However, to go down to future generations, engineered bacteria have the stability of plasmid and protein yield to a large extent. The amount of the fusion protein was also up to 40% of the total cell protein after the 50th generation. These data would be useful for the industrial production of the recombinant GnRH vaccine.This work was supported by the National High Technology “863” Programs of China (no. 2002 AA217031-2), a Grant-in-Aid from China National Natural Science Fund Committee (grant no. 30270298) and Jiangsu Natural Science Fund Committee (grant no. BK 95092309 and BG2001011).     

奥利亚罗非鱼促性腺激素释放激素前体蛋白的克隆与表达

促性腺激素释放激素（gonadotropin—relying hormone,GnRH）是下丘脑分泌产生的神经激素,对脊椎动物生殖的调控起重要作用。为研究GnRH对性腺发育的作用,构建了GnRH cDNA的原核表达载体并进行融合表达。利用RT—PCR方法从奥利亚罗非鱼丘脑中扩增出长约400bp的目的序列GnRH基因,克隆至T载体中,经酶切鉴定和序列测定分析确认序列的正确性后将此片段克隆到表达载体pMAL—c2x中构建重组表达质粒pMAL—GnRH,并在大肠杆菌TB1中获得了高表达,目的蛋白约占菌体总蛋白的41．6％。菌体经溶菌酶裂解,制备无细胞抽提液,Amylose—sepharose柱层析后得到分子量为56kD单一条带的目的蛋白。目的蛋白经Factor Xa酶切裂解,Amylose—sepharose过柱纯化后得到纯化的GnRH前体蛋白。该研究为鱼类GnRH蛋白的控制性腺成熟和抗体制备打下了基础．是国内鱼类GnRH前体蛋白在原核细胞中成功表扶的首次报道．     

奥利亚罗非鱼促性腺激素释放激素cDNA的原核表达及其免疫原性研究

促性腺激素释放激素(gonadotropin-releasinghormone,GnRH)是下丘脑分泌产生的神经激素,对脊椎动物生殖的调控起重要作用。为研究GnRH对奥利亚罗非鱼性腺发育的作用,构建了GnRHcDNA的原核表达载体并进行融合表达。利用RT-PCR方法从丘脑中扩增出长约400bp的目的序列GnRH基因,克隆至T载体中,经酶切鉴定和序列测定分析确认序列的正确性后将此片段克隆到表达载体pMAL-c2x中构建重组表达质粒pMAL-GnRH,并在大肠杆菌TB1中获得了高表达,目的蛋白约占菌体总蛋白的41.6%。菌体经溶菌酶裂解,制备无细胞抽提液,Amylose-sepharose柱层析后得到分子量为56kD单一条带的目的蛋白。目的蛋白经FactorXa酶切裂解,Amylose-sepharose过柱纯化后得到纯化的GnRH前体蛋白。以80μg/只的剂量4次免疫ICR小鼠,免疫小鼠可以检测到特异性针对GnRH前体蛋白的血清抗体应答,免疫组抗体水平显著高于空白组(P<0.05),且加强免疫第5周后抗体效价为0.707±0.320,达到高峰值,说明表达产物具有免疫原性,可以刺激机体产生免疫应答。     

Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin

An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.     

Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+ -ATPase

A Ca2+ -dependent calmodulin-binding peptide (CBP) is an attractive tag for affinity purification of recombinant proteins, especially membrane proteins, since elution is simply accomplished by removing/chelating Ca2+. To develop a single-step calmodulin/CBP-dependent purification procedure for Escherichia coli nicotinamide nucleotide transhydrogenase, a 49 amino acid large CBP or a larger 149 amino acid C-terminal fragment of human plasma membrane Ca2+ -ATPase (hPMCA) was fused C-terminally to the beta subunit of transhydrogenase. Fusion using the 49 amino acid fragment resulted in a dramatic loss of transhydrogenase expression while fusion with the 149 amino acid fragment gave a satisfactory expression. This chimeric protein was purified by affinity chromatography on calmodulin-Sepharose with mild elution with EDTA. The purity and activity were comparable to those obtained with His-tagged transhydrogenase and showed an increased stability. CBP-tagged transhydrogenase contained a 4- to 10-fold higher amount of the alpha subunit relative to the beta subunit as compared to wild-type transhydrogenase. To determine whether the latter was due to the CBP tag, a double-tagged transhydrogenase with both an N-terminal 6x His-tag and a CBP-tag, purified by using either tag, gave no significant increase in purity as compared to the single-tagged protein. The reasons for the altered subunit composition are discussed. The results suggest that, depending on the construct, the CBP-tag may be a suitable affinity purification tag for membrane proteins in general.     

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.     

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for ¹⁵N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.     

GnRH-PEⅡ-luffinS重组嵌合毒素的制备与细胞毒性检测

目的：制备以Ⅰ型促性腺激素释放激素（GnRH Ⅰ）为导向部分,以绿脓杆菌外毒素的结构域Ⅱ（PEⅡ）为转膜区,以丝瓜毒素luffinS2为毒性部分的重组嵌合毒素GnRH-PEⅡ-luffinS,体外实验检测其对肿瘤细胞的杀伤作用。方法：重叠PCR法扩增GnRH-PEⅡ-luffinS的基因,克隆至表达载体pET32a中,转化大肠杆菌BL21(DE3),挑取阳性克隆诱导表达,产物用Ni-NTA亲和层析柱纯化。纯化蛋白经重组肠激酶（rEK）切割去除Trx融合蛋白,XTT法检测重组毒素对HeLa、A549、HepG-2、SP2/0和鸡胚成纤维细胞（CEF）的体外细胞毒作用。结果：成功构建了GnRH-PEⅡ-luffinS的表达质粒,并在大肠杆菌中获得表达,纯化后的纯度为94％。GnRH-PEⅡ-luffinS对HeLa、A549、HepG-2和SP2/0的IC50分别为13.50μg/ml、13.74μg/ml、16.79μg/ml和26.07μg/ml,而对CEF无作用。结论：重组嵌合毒素GnRH-PEⅡ-luffinS对肿瘤细胞有较强的杀伤作用。     

hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究

为进一步改造重组人激肽释放酶1(hK1),以期提高其生物活性,制备了通过柔性接头相连接的重组人激肽释放酶1-L-IgG1 Fc融合蛋白(hK1-L-Fc)。采用重叠延伸PCR技术构建hK1-L-Fc融合基因,克隆至表达载体pcDNA3.1,在中国仓鼠卵巢细胞(CHO-S)中表达。利用Protein A 亲和层析柱纯化融合蛋白,SDS-PAGE、Western blotting、飞行时间质谱(MALDI-TOF-MS)、HPLC检测表达产物,底物法检测融合蛋白的体外活性。结果显示:成功构建pcDNA3.1-hK1-L-Fc重组表达载体;获得稳定表达融合蛋白的细胞株;无血清悬浮批式培养的表达量在0.7 mg/L以上;纯化的蛋白其纯度在95%以上,分子量约60 kDa;活性检测显示其比活性在9.2 U/mg以上,较hK1-Fc蛋白提高了18%以上。     

High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli

The hybrid antibacterial peptide CA-MA [cecropinA(1-8)-magainin2(1-12)] with potent antimicrobial properties but no hemolytic activity is a potential alternative antibiotic. To explore a new approach for high-level expression of the hybrid peptide CA-MA in Escherichia coli, the sequence of ubiquitin (UBI) from housefly was inserted into the plasmid pQE30 to construct the vector pQEUBI. The cDNA fragment encoding CA-MA with preferred codons of E. coli was obtained by recursive PCR (rPCR) and cloned into the vector pQEUBI to express the fusion protein (His)(6)-UBI-CA-MA. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 36% of the total proteins). With (His)(6)-tag, the fusion protein was easily purified by Ni-NTA chromatography and 36 mg of fusion protein was purified from 1L of culture medium. The fusion protein was efficiently cleaved by ubiquitin C-terminal hydrolase (UCH), yielding recombinant CA-MA with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant CA-MA was purified to homogeneity by reversed-phase HPLC and 6.8mg of pure active CA-MA was obtained from 1L culture medium. Analysis of recombinant CA-MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA-MA was 2559Da, which perfectly matches the mass (2559Da) calculated from the amino acid sequence. Analysis of CA-MA by circular dichroism (CD) revealed that the secondary structures of CA-MA in water solution were 17.4% alpha-helix and 82.6% random coil but no beta-sheet. Our results demonstrated that functional CA-MA can be produced in sufficient quantities using the ubiquitin fusion technique. This is the first report on the heterologous expression of a hybrid antibacterial peptide fused to ubiquitin in E. coli.     

Recombinant Production, Isotope Labeling and Purification of ENOD40B: A Plant Peptide Hormone

The plant peptide hormone ENOD40B was produced in a protein production strain of Escherichia coli harboring an induction controller plasmid (Rosetta(DE3)pLysS) as a His6-tagged ubiquitin fusion protein. The fusion protein product was denatured and refolded as part of the isolation procedure and purified by immobilized metal ion chromatography. The peptide hormone was released from its fusion partner by adding yeast ubiquitin hydrolase (YUH) and subsequently purified by reversed phase chromatography. The purity of the resulting peptide fragment was assayed by MALDITOF mass spectrometry and NMR spectroscopy. The final yields of the target peptide were 7.0 mg per liter of LB medium and 3.4 mg per liter of minimal medium.     

黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 kD,对革兰氏阳性菌(G+)和革兰氏阴性菌(G?)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体pET22b-ELP20-EK-YFGAP、pET22b-ELP40-EK-YFGAP和pET22b-ELP40-L-EK- YFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。     

Production of biologically active thymulin in Escherichia coli through expression of a chemically synthesized gene

Summary A synthetic gene coding for thymulin was ligated into an expression vector (pJB 1301) and placed under lac operon control. In the recombinant clones, thymulin was expressed as part of a galactosidase chimeric protein which was then cleaved by cyanogen bromide. Thymulin was purified using various chromatography systems including gel filtration and HPLC, and was detected by radioimmunoassay (RIA). In the biological assay the purified recombinant peptide demonstrated the same zinc dependency as natural thymulin and had the same amino acid composition and primary structure.     

一种抗菌肽和aFGF融合蛋白的构建和表达

利用PCR技术扩增出带有凝血酶Xa因子切割位点的天蚕素蜂毒素杂合肽和aFGF的融合基因,插入大肠杆菌表达载体pET-3c中,构建出表达质粒pET-aF-CM,并转化至大肠杆菌BL21（DE3）中,氨苄青霉素抗性筛选重组转化子。IPTG诱导4h后,以包涵体形式表达的融合蛋白约占菌体总蛋白的17%。将包涵体溶解后透析复性,并利用肝素亲和层析纯化,得到电泳纯的融合蛋白。Western blot分析表明,该蛋白能与aFGF抗体产生免疫反应。MTT法检测显示,融合蛋白具有促3T3Bal/b细胞分裂活性,其比活为1.471×106IU/mg。利用凝血酶Xa因子裂解融合蛋白,可以获得抗菌肽和含凝血酶Xa因子裂解序列的aFGF蛋白。分子筛回收含杂合抗菌肽,抑菌活性检测表明其对大肠杆菌K12D31具有明显抑菌活性。微量稀释法检测结果表明,回收的抗菌肽对大肠杆菌DH5α、大肠杆菌K12D31、沙门氏菌、金黄色葡萄球菌、枯草芽孢杆菌和绿脓杆菌的MIC分别达6.25μg/ml、10μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml和5μg/ml。     

旋毛虫肌幼虫ES抗原的基因克隆及高效表达

作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0．7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。     

人CS1-Fc融合蛋白的真核表达、蛋白纯化及生物学效应鉴定

人信号淋巴细胞激活分子F7 (SLAMF7/CS1)是一种细胞表面糖蛋白,在多发性骨髓瘤细胞中高度表达。已有研究表明CS1是多发性骨髓瘤较为灵敏且特异的生物标志物。CAR-T细胞免疫疗法是治疗多发性骨髓瘤的新方法,其中CS1 CAR-T细胞免疫疗法针对复发性难治性多发性骨髓瘤有较好的疗效。为了检测CS1 CAR-T细胞上CS1CAR的表达效率和探寻CAR-T细胞免疫疗法的辅助手段,文中制备了一种CS1-Fc融合蛋白。首先利用PCR技术从已有质粒中扩增得到CS1的胞外段序列,再通过重叠延伸PCR与人IgG1-Fc段相连。将重组片段连接至pMH3真核表达载体上,经酶切鉴定和DNA测序后,将重组质粒pMH3-CS1-Fc-his转染至中国仓鼠卵巢细胞(CHO-S)。经G418加压筛选和流式细胞术鉴定,证实CS1-Fc融合蛋白在CHO-S细胞中获得了表达。利用镍柱对CS1-Fc融合蛋白进行纯化,经Western blotting鉴定,融合蛋白的分子量约为70 kDa。流式细胞术和细胞计数分析结果显示,CS1-Fc融合蛋白能有效检测CS1 CAR的表达效率,证实了CS1-Fc融合蛋白对CS1 CAR-T细胞具有活化、促增殖及促分泌细胞因子的作用。本研究结果为多发性骨髓瘤细胞免疫治疗CAR-T细胞的体外检测和效能强化奠定了实验基础。     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.     

Preparation and characterization of the recombinant protein containing immunomimetic peptide of benzo[a]pyrene

Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor

The specific molecular interactions of alpha-helical peptide, human glucagon (i.e., intermolecular self-association and specific receptor-binding affinity) provided a rationale for using the glucagon as the fusion expression partner to achieve high productivity of foreign proteins both in vivo (in bacterial fusion-expression system) and in vitro (in affinity column chromatography). The fusion of glucagon peptide(s) effectively promoted homogeneous aggregate formation of recombinant proteins while avoiding intermolecular crosslinking by disulfide bridges. High sensitivity of the self-aggregation to sequence effects resulted from two distinct nonpolar domains of glucagon, determining specificity of molecular interaction and aggregate size of recombinant proteins. An N-terminal domain of glucagon molecule (Phe6-Tyr10-Tyr13) could be a certain hydrophobic moiety involved in intermolecular self-association (probably, via helix-helix docking), while a C-terminal domain (Phe22-Trp25-Leu26) seems to critically affect the oligomer size in the off-pathway aggregation of synthesized fusion proteins. An N-terminal extracellular domain of human glucagon receptor was recombinantly expressed in Escherichia coli, immobilized to a chromatography column, and efficiently renatured to a conformation that attains high specificity in interaction with N-terminus glucagon molecules of recombinant fusion proteins. Through column chromatography employing the receptor fragment as affinity ligand, the recombinant proteins were efficiently purified from total intracellular proteins, and the long-term ligand stability was evidently proven through multiple cyclic-purification experiments. Major scaffolds for using protein ligands are large-scale production in a low-cost expression system and long-term stable operation with selective-binding affinity. From this point of view, the extracellular fragment of human glucagon receptor used in this study seems to be a new potent ligand for fusion protein-based affinity chromatography.