人胰岛素原基因在酵母中的分泌表达

 我们研究了外源基因——合成的人胰岛素原基因在酵母α因子系统中的表达和分泌。用表达载体YTI-15转化酵母63号株可得到每升约2毫克的人胰岛素原分泌产物。     

A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single-chain des-(b-30)-lnsulin precursor with lysyl endopeptidase

It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobacter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.     

Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)     

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Key words proinsulin; C-peptide; Pichia pastoris     

胰岛素原C肽多聚体基因在大肠杆菌中的表达及重组C肽在水溶液中的稳定性

合成了胰岛素原C肽三聚体的基因,并在大肠杆菌中得到高效表达。表达的融合蛋白质通过设计的特异性位点的酶切得到重组的人胰岛素原C肽单体。融合蛋白质以可溶性蛋白质的形式表达,表达量约为80mg／L。Ni—NTA亲和柱可有效地从细胞裂解的上清液中分离纯化融合蛋白质,得到纯度大于70％的融合蛋白质37．5mg／L。融合蛋白质可经胰蛋白酶/羧肽酶B双酶切有效释放天然的C肽。释放的C肽经氨基酸组成分析、与标准对照的RP—HPLC分析和免疫发光法定量证实与天然人C肽完全相同。C肽经RP-HPLC纯化后可得,总的收率为1．5mg/L,纯度大于95％。考察了C肽在冻干过程中及在水溶液中的稳定性。结果表明C肽在冻干过程中稳定,在水溶液中其稳定性受pH和温度的影响。在pH3和pH7．4缓冲液中,37℃或70℃下,C肽的降解符合一级动力学。C肽冻干品直接溶解于pH9的缓冲液中,立即降解,降解产物在37℃和70℃下基本稳定。C肽冻干品直接溶解于pH3的缓冲液中,立即发生降解反应,随后可观察到随温度升高和时间延长,降解反应的进行,37℃、10h,可观察到约80．3％的C肽保持,而在70℃、10h,只有43％C肽保持。C肽在pH7．4最稳定,37℃、6h或10g／L BSA存在下,70℃、3h,未观察到降解产物。PH7．4、37℃、10h,在有和没有10g／L BSA存在的情况下,C肽的保持率分别为99％和96％,从而显示了BSA的保护作用。     

hGH-双精氨酸C肽人胰岛素原基因的克隆表达及纯化研究

旨在提高基因重组人胰岛素在大肠杆菌中表达的稳定性及表达包涵体蛋白的复性水平.在人胰岛素原N端前融合人生长素N端的一段序列来充当前导肽,同时将C肽设计为两个精氨酸,分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增得到该基因片段.与表达载体PET-30a连接,转化E.coli BL21(DE3),IPTG诱导表达.表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性、冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再过DEAE Sepharose Fast Flow阴离子交换柱,收集洗脱峰.对制备所得的胰岛素用SDS-PAGE,Western blot进行性质鉴定,及皮下注射小鼠测定生物活性.结果显示,目的蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%.经Ni-NTA亲和层析得到的重组蛋白纯度为85%,DEAE Sepharose Fast Flow阴离子交换纯化得到单组分胰岛素.Western Blot显示制备所得的胰岛素具有胰岛素免疫原性,皮下给药注射小鼠活性测定表明具有明显的降血糖活性.获得了一种高效生产基因重组人胰岛素的方法,为研究胰岛素类似物奠定了前期基础同时也为今后探索胰岛素的非注射给药途径提供了原料.     

Characterization of Antibodies to Products of Proinsulin Processing Using Immunofluorescence Staining of Pancreas in Multiple Species

The efficient processing of proinsulin into mature insulin and C-peptide is often compromised under conditions of beta cell stress, including diabetes. Impaired proinsulin processing has been challenging to examine by immunofluorescence staining in pancreas tissue because the characterization of antibodies specific for proinsulin, proinsulin intermediates, processed insulin and C-peptide has been limited. This study aimed to identify and characterize antibodies that can be used to detect products of proinsulin processing by immunofluorescence staining in pancreata from different species (mice, rats, dog, pig and human). We took advantage of several knockout mouse lines that lack either an enzyme involved in proinsulin processing or an insulin gene. Briefly, we report antibodies that are specific for several proinsulin processing products, including: a) insulin or proinsulin that has been appropriately processed at the B-C junction; b) proinsulin with a non-processed B-C junction; c) proinsulin with a non-processed A-C junction; d) rodent-specific C-peptide 1; e) rodent-specific C-peptide 2; and f) human-specific C-peptide or proinsulin. In addition, we also describe two ‘pan-insulin’ antibodies that react with all forms of insulin and proinsulin intermediates, regardless of the species. These antibodies are valuable tools for studying proinsulin processing by immunofluorescence staining and distinguishing between proinsulin products in different species.     

从基因工程小C肽人胰岛素原类似物（B－R2－A）制备人胰岛素

以融合蛋白的形式,在Ｅ．ｃｏｌｉ中经温度诱导表达了小Ｃ肽人胰岛素原类似物（Ｂ－Ｒ２－Ａ）．表达的融合蛋白可占细胞总蛋白６８％．经磺酸解,及初步分离Ｓ－磺酸型融合蛋白,再经ＣＮＢｒ裂解后,进行还原重组,ＨＰＬＣ分离纯化等步骤,每升发酵液可得到Ｂ－Ｒ２－Ａ约５０ｍｇ。经酶促转化及ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５纯化,得重组人胰岛素约２０ｍｇ,其氨基酸组成与人胰岛素相同,并具有与猪胰岛素相同的生物活性．     

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line,CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.