Saima Sadaf

Production of bubaline somatotropin by auto-induction in Escherichia coli

Production of His–BbST [hexahistidine‐tagged BbST (bubaline somatotropin)] by auto‐induction in high‐density shake‐flask cultures coupled with a single‐step, on‐column purification and refolding strategy is described here. To optimize expression of BbST, different media and expression conditions were tested. The highest expression levels of BbST, exceeding 30% of the total Escherichia coli cellular proteins, were achieved when YNG and M9NG media were used. Using these auto‐inducing media, the final concentration of BbST increased up to 455 mg/l and was severalfold higher than that obtained by isopropyl β‐d‐thiogalactoside induction. Most of the target protein, however, was in the form of inclusion bodies, which were solubilized in 8 M urea solution (pH 8.5). Using immobilized‐metal‐ion‐affinity chromatography, His–BbST could be purified from solubilized sample to >97% homogeneity in a single step in a biologically active state as judged by its efficient growth‐promoting activity in Nb2 rat lymphoma cell proliferation assays. The expression and purification scheme, presented here, has a potential of scaling up to obtain pure and biologically active His–BbST relatively inexpensively for further studies and applications.

Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli

A putative β-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-β-D-1-thiogalactopyranoside, the enzyme expressed at ∼40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A600 12 in auto-inducing M9NG medium, β-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the β-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45°C and 8.0, respectively, under the assay conditions. Km and kcat against p-nitrophenyl-β-D-glucopyranoside were 4 mM and 0.75 sec−1, respectively. To our knowledge, this is the first report of high-level expression and characterization of a β-glucosidase from B. halodurans.

Role of silent gene mutations in the expression of caprine growth hormone in Escherchia coli.

This report describes the strategy for overexpression of caprine growth hormone (cGH) gene of beetal goat in E. coli through introducing silent mutations in the 5′‐end of the coding sequence. The silent mutations introduced were aimed at minimizing translation‐inhibiting secondary structures in the mRNA. Free energies of the resultant mRNAs were calculated from the ribosomal binding site of mRNA to +24 base using the Mfold web server. The construct with native sequence did not show any expression, whereas introduction of the silent mutations had strong influence on the expression levels. Some constructs (pETcGH2–7) showed 12–30% expression of total cell proteins while some others (pETcGH8–16) showed 30 to 53% of total cell protein. Any variation in the amount of mRNA transcript for the various constructs, as determined by quantitative PCR, was not enough to suggest that the variable level of the gene expression was due to variation in the transcription levels. It appears that the expression levels are not always correlated with free‐energy values of the secondary structures in the 5′‐end region of the mRNA; instead some key silent nucleotide alterations at certain sites of 5′‐end of the sequence reorganize the secondary structure in such a way that it has positive impact on translation without considerably altering the free‐energy values. An empirical approach for determining the optimum 5′‐end substitutions for hyperexpression of a recombinant protein thus seems necessary.

Production enhancement and refolding of caprine growth hormone expressed in Escherichia coli.

This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2-3 times higher cell densities were obtained in shake flask cultures when induction was done with lactose showing almost same level of expression as in case of IPTG induction. With lactose induction highest cell densities were achieved in TB (OD(600) 16.3) and M9NG (OD(600) 16.1) media, producing 885 and 892 mg cGH per liter of the culture, respectively. Lactose induction done at mid-exponential stage resulted in a higher cell density and thus higher product yield. cGH over-expressed as inclusion bodies was solubilized in 50 mM Tris-Cl buffer (pH 12.5) containing 2 M urea, followed by dilution and lowering the pH in a step-wise manner to obtain the final solution in 50mM Tris-Cl (pH 9.5). The cGH was purified by Q-Sepharose chromatography followed by gel filtration with a recovery yield of 39% on the basis of total cell proteins. The product thus obtained showed a single band by SDS-PAGE analysis. MALDI-TOF analysis showed a single peak with a mass of 21,851 dalton, which is very close to its calculated molecular weight. A bioassay based on proliferation of Nb2 rat lymphoma cells showed that the purified cGH was biologically active.

Molecular cloning, characterization, and expression studies of water buffalo (Bubalus bubalis) somatotropin

Cloning, high-level expression, and characterization of the somatotropin (ST) gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis (BbST) are described. Coding, non-coding, and promoter regions of BbST were amplified and sequenced. Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species. One interesting variation in the BbST sequence was the replacement of a conserved glutamine residue by arginine. A plasmid was also constructed for the production of BbST in Escherichia coli BL21 (RIPL) CodonPlus, under the control of IPTG-inducible T7-lac promoter. High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies. The inclusion bodies represented over 20% of the E. coli cellular proteins. The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay. The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure—function studies and livestock industry.

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6.

This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer.

Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using auto- inducing medium

A subtilisin gene identified in the reported genome sequence of Pyrococcus furiosus was amplified and inserted in pET-22b(+) vector to produce the recombinant plasmid pET-SB. Escherichia coli BL-21 (DE3) CodonPlus was transformed with this plasmid and the enzyme was expressed up to 30% of the total cell protein on induction with IPTG. The expressed protein appeared at a position corresponding to ~20 kDa on SDS-PAGE as compared to theoretical molecular mass of 17.6 kDa. This aberrant electrophoresis mobility could be due to specific amino acid composition of the protein. Auto-induction with lactose also produced a similar level of expression but the total amount of the enzyme produced was 2.4 fold greater than that when produced with IPTG induction. This was due to a higher cell density obtainable in the auto-inducing medium. The enzyme expressed in the insoluble state could be partially refolded after denaturation with urea at high pH. This study reports for the first time high-level expression of subtilisin of P. furiosus in E. coli using an auto-inducing medium.

Identification of actin beta-like 2 (ACTBL2) as novel, upregulated protein in colorectal cancer

Early diagnosis of colorectal cancer (CRC) can be of value for increasing the survival rate of patients. Recently, proteomic strategies to identify markers for the diagnosis of cancer at an early stage have been employed with noteworthy results. To extend these studies, we utilized two dimensional gel electrophoresis and mass spectrometry for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls). Four gel spots were determined to be differentially stained between the tumor and the control samples on a consistent basis. Following mass spectrometric analysis of these spots, six proteins were identified; five of these had previously been reported to be associated with colorectal cancer. One protein actin beta-like 2 (ACTBL2), not linked with colorectal cancer in the earlier reports, was however found to be at higher abundance in colorectal tumor samples both by proteomics and immunohistochemistry analysis. Thus ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer. BIOLOGICAL SIGNIFICANCE Colorectal cancer (CRC) is a major cause of death world-wide and good markers for early detection are lacking. In this study we conducted a proteomic analysis of tumor vs. normal tissue. We corroborated the finding of a number of previously identified proteins associated with CRC and more importantly identified a novel protein, ACTBL2, which we demonstrated to be upregulated in CRC. As additional proteins associated with CRC are identified the potential for developing panels of markers may be realized with better outcomes in early cancer detection.

Synonymous codon changes at the 5′-end of the gene strongly impact the heterologous protein expression in Escherichia coli

This study describes the impact of 5′-end codon modulation on the expression of a heterologous gene, human granulocyte colony stimulating factor (GCSF), in Escherichia coli. Fourteen different constructs (pGCSF-01 to pGCSF-14) carrying single or multiple synonymous substitutions at +2, +3 and further down from +4 to +7 codons, were prepared and their expression was monitored in E. coli BL21 Codon-Plus (DE3) RIPL using a strong T7 lac-promoter based expression system. A single nucleotide change at +2 Thr codon (ACC→ACA) either alone or in combination with +3 Pro codon (CCC/CCT/CCA) resulted in the expression enhancement of an otherwise poorly expressed native-GCSF, to a level that corresponded to 45–50% of the total E. coli BL21 CodonPlus (DE3) RIPL cellular proteins. The differences in GCSF expression amongst different constructs could be attributed to the preferential or non-preferential codon usage, reduced number of G/C nucleotides and the stability of mRNA secondary structure formed near the 5′-end coding region. The expression of GCSF achieved was in the form of biologically inactive inclusion bodies that were solubilized using mild concentration of a non-ionic surfactant and refolded by a simplified, step-dialysis approach. Biological activity of the purified GCSF, assessed in induced neutropenic mice, was similar to the commercially available preparation of the GCSF analog (filgrastim).

Expression and sequence characterization of growth hormone binding protein of Nili-Ravi buffaloes ( Bubalus bubalis )

The growth hormone binding protein (GHBP) was isolated from the liver of Nili-Ravi buffaloes ( Bubalus bubalis ), reverse transcriptase-polymerase chain reaction (RT-PCR) amplified and sequence characterized. RT-PCR analysis demonstrated high degree sequence identities (97.3 to 99.6%) of Bb GHBP cDNA with Bos taurus , Ovis aries and Capra hircus . An expression plasmid was constructed for the production of Bb GHBP in Escherichia coli BL21 (RIPL) CodonPlus under the control of T7lac promoter. On induction with isopropyl β-D thiogalactopyranoside, the Bb GHBP was expressed at levels >30% of the total E. coli proteins. The target protein expressed as inclusion bodies was solubilized in denaturing solution and refolded by step/pulsatile dilution method using cysteine and cystine redox potential. Purification to near homogeniety (>98%) was achieved by ion-exchange chromatography with a recovery yield of 64%. Mass spectrometric analysis of the purified Bb GHBP showed a single peak of 30,756 Da. A radioprotein assay evaluated the binding affinity of recombinant Bb GHBP with iodinated bovine growth hormone (bGH) which demonstrated active conformation of Bb GHBP. These results demonstrate high expression and sequence characterization of Bb GHBP in Nili-Ravi buffaloes and provide the basis for the assessment of Bb GHBP in other breeds of buffalo. Keywords: Liver, Nili-Ravi buffalo, GHBP, MALDI-TOF mass spectrometry, radioprotein binding assay, refolding