Cloning a synthetic gene for human stefin B and its expression in E. coli

A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with beta-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.     

Inhibitorily active recombinant human stefin B. Gene synthesis, expression and isolation of an inhibitory active MS-2 pol-stefin B fusion protein and preparation of Des[Met1,2(2)]stefin B

A synthetic gene coding for the human intracellular cysteine proteinase inhibitor, stefin B, was constructed from 13 chemically synthesized oligonucleotides according to the method of Khorana. The gene was inserted into the plasmid vector pTZ, amplified and sequenced. For expression, a temperature-inducible system producing fusion proteins was used. With the vector pEx31A containing the synthetic cystatin B gene, E. coli strain 537 produced a fusion protein of the N-terminal part of bacteriophage MS-2 polymerase and [Met-2Gly-1]stefin B. Lysates of the induced bacteria were inhibitorily active against papain. The fusion protein was expressed in high yield (about 20% of total E. coli proteins) and mostly deposited as inclusion bodies. The unfolded fusion protein was partially purified in the presence of urea. After refolding, approx. 6% of the protein was inhibitorily active against papain, human cathepsin H and B. Des[Met1,2(2)]stefin B was released by cyanogen bromide cleavage of the fusion protein and identified by N-terminal amino-acid sequence analysis. The non-separated cleavage products were also inhibitorily active after refolding. The estimated inhibition constants for the fusion protein and its cleavage products were similar to those reported for natural stefin B.     

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.     

Periplasmic secretion of human growth hormone by Escherichia coli

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.     

Isolation of the Actinobacillus pleuropneumoniae haemolysin gene and the activation and secretion of the prohaemolysin by the HlyC, HlyB and HlyD proteins of Escherichia coli

The gene encoding the c. 105 kD secreted haemolysin protein of the porcine pathogen Actinobacillus pleuropneumoniae serotype 1 has been isolated by screening a lambda gt11 expression library in Escherichia coli with antiserum raised against the wild-type protein. A derivative recombinant DNA pJFF702 expressed the hlylA haemolysin gene from the pUC19 lac promoter but the resulting haemolysin I protein remained within the E. coli cell and was haemolytically inactive. Export of the intracellular A. pleuropneumoniae prohaemolysin out into the medium was achieved by the presence in trans of the E. coli haemolysin secretion genes hlyB and hlyD, and high levels of intracellular haemolytic activity were attained similarly by the E. coli post-translational haemolysin activator gene, hlyC. Southern hybridization of A. pleuropneumoniae parental DNA nevertheless indicated only a low degree of nucleotide sequence identity to the haemolysin structural and secretion genes hlyA and hlyB of E. coli. The data show that despite substantial nucleotide sequence divergence the A. pleuropneumoniae serotype 1 haemolysin determinant is closely related to that which is dispersed throughout other Gram-negative human and animal pathogens.     

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.     

Simultaneous synthesis of human-, mouse- and chimeric epidermal growth factor genes via ''hybrid gene synthesis'' approach.

Simultaneous synthesis of two DNA duplexes encoding human and mouse epidermal growth factors (EGF) was accomplished in a single step. A 174 b.p. DNA heteroduplex, with 16 single and double base pair mismatches, was designed. One strand encoded the human EGF, and the opposite strand indirectly encoded the mouse EGF. The heteroduplex DNA was synthesized by ligation of seven overlapping oligodeoxyribonucleotides with a linearized plasmid. After transformation in E. coli HB101 (recA 13), the resulting heteroduplex plasmid served as the template in plasmid replication. Two different plasmid progenies bearing either the human or mouse EGF-coding sequence were identified by colony hybridization using the appropriate probes. However, in E. coli JM103, the same process yielded plasmid progenies encoding different chimeric EGF molecules, presumably due to crossover of human and mouse EGF gene sequences.     

重组人干扰素α-2b在大肠杆菌中分泌表达

人干扰素α-2b原始基因在重组原核工程菌中表达量偏低,所以我们在不改变干扰素原有氨基酸组成的前提下,根据大肠杆菌密码子偏爱性使用定向突变技术对huIFNα-2b基因进行点突变。将大肠杆菌STⅡ信号肽基因与突变后huIFNα-2b基因融合并于信号肽5′端和huIFNα-2b基因3′端引入合适的酶切位点。融合基因克隆至载体pCSE,pET-22b和pPAK4L中,此3种载体分别含有组成型启动子、T7启动子和phoA启动子。融合基因在载体pCSE中表达量很低,其中约有50%的目标蛋白能够成功实现分泌。在E.coliBL21中,pET-22b经过IPTG诱导可以实现huIFNα-2b的高表达,但STⅡ信号肽不能被有效切除。含有phoA启动子的载体pPAK4L其在E.coliW3110中可以实现huIFNα-2b较高水平的分泌表达,经过低磷诱导其表达量最高可至20μg/mL(A550)菌液,约有30%的目标蛋白质信号肽能够被成功切除并分泌到胞间质中。     

Structure and properties of a human non-pancreatic phospholipase A2

We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.     

Secretory production of human leptin in Escherichia coli

Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.     

Secretion of recombinant proteins into the culture medium by Escherichia coli and Staphylococcus aureus

The ability of staphylococcal protein A (SPA) to bind to the Fc part of IgG has been used for the purification of a number of heterologous gene products as fusion proteins. Both the SPA promoter and signal sequence function in Escherichia coli, as well as in a number of Gram-positive bacteria, which facilitates comparisons of the expressed specific products in different hosts. The expression system developed for E. coli yields excretion of the fusion protein to the growth medium, which makes E. coli a competitive alternative to Gram-positive bacteria for the expression of secreted products. The human peptide hormones insulin-like growth factors (IGF) I and II were expressed using the protein A system in E. coli and Staphylococcus aureus. Despite a high degree of structural homology, large differences in the yields were observed in the two hosts. This underlines the importance of investigating different bacterial hosts for a particular protein product.     

Bacillus licheniformis6816碱性蛋白酶基因在E.coli中的克隆和表达

用PCR方法从地衣芽孢杆菌6816中扩增了碱性蛋白酶基因（apr),扩增的1.14kb的DNA片段插入到大肠杆菌载体pET-20b中,构建成重组分泌型表达载体pAPR1。pAPR1中碱性蛋白酶基因在大肠杆菌宿主JM109（DE3）中得到表达,SDS-PAGE分析显示融合表达产物的分子量为30kD,同核酸序列测定所推导的值相符,表达产物占细胞总蛋白的7.5%,重组菌的酶活比出发菌株提高了3.3倍,研究发现,重组的碱性蛋白酶在进入大肠杆菌周质空间时存在前肽自动脱落的现象。     

Secretion of Bacillus subtilis alpha-amylase in the periplasmic space of Escherichia coli

The Bacillus subtilis alpha-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis alpha-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.     

Recombinant anti-stefin A Fab fragment: sequence analysis of the variable region and expression in Escherichia coli

Human stefin A is an inhibitor of lysosomal cysteine proteinases cathepsin B, H, L and S. In the present report we describe the cloning and expression of anti-stefin A Fab fragment A22 in E. coli. We have determined the nucleotide sequences of the antibody heavy and light chain and compared them to the murine immunoglobulin germ line sequences. Expression of the two antibody chains was achieved using a single vector with a PhoA promoter and coding regions placed after the signal sequences, directing them to the periplasmic space. The A22 Fab fragment was extracted from the periplasmic space and expression was confirmed by Western blot analysis. The recombinant A22 Fab fragment had an affinity for stefin A comparable to the original monoclonal antibody, as determined by ELISA.     

Chemical Synthesis of Proteinase Inhibitor Genes

Abstract

Genes coding for the two human cysteine proteinase inhibitors (cystatin C, stefin A) were obtained by enzymic ligation of chemically synthesized oligodeoxynucleotides. This article reports the results from the expression of these proteinase inhibitor genes in E. coli and from the isolation and characterization of the corresponding proteins.     

Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein)

A new secretion vector, pEAP84 which contained a unique restriction site (BglII) at the 3' end of the penicillinase gene to produce a fused protein, and the Ex-kil region to make the outer membrane permeable, was constructed from pEAP82. A recombinant plasmid p84h06, which contained a synthetic gene for human calcitonin with a cyanogen bromide cleavage site at the junction site of the fused protein, was constructed and introduced into Escherichia coli. The hybrid protein produced in E. coli carrying p84h06 was secreted into the culture medium. The amino acid composition of this product was consistent with that deduced from the DNA sequence. Mature calcitonin was obtained following cyanogen bromide cleavage of the fused protein.     

The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium

A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a lambda gt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.