Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region.

BACKGROUND: High thermostability is required for trypsin to have wider industrial applications. Target mutagenesis at flexible regions has been proved to be an efficient protein engineering method to enhance the protein thermostability. RESULTS: The flexible regions in porcine trypsin were predicted using the methods including molecular dynamic simulation, FlexPred, and FoldUnfold. The amino acids 78-90 was predicted to be the highly flexible region simultaneously by the three methods and hence selected to be the mutation target. We constructed five variants (D3, D5, D7, D9, and D11) by truncating the region. And the variant D9 showed higher thermostability, with a 5 °C increase in Topt, 5.8 °C rise in [Formula: see text], and a 4.5 °C rise in Tm, compared to the wild-type. Moreover, the half-life value of the variant D9 was also found to be dramatically improved by 46 min. Circular dichroism and intrinsic fluorescence indicated that the structures had no significant change between the variant D9 and the wild-type. The surface hydrophobicity of D9 was measured to be lower than that of wild-type, indicating the increased hydrophobic interaction, which could have contributed to the improved thermostability of D9. CONCLUSIONS: These results showed that the thermostability of variant D9 was increased. The variant D9 could be expected to be a promising tool enzyme for its wider industrial applications. The method of truncating the flexible region used in our study has the potential to be used for enhancing the thermostability of other proteins.

Isoform separation and structural identification of mono-PEGylated recombinant human growth hormone (PEG-rhGH) with pH gradient chromatography.

Human growth hormone plays an essential role in the treatment of dwarfism diseases, but it is limited in its short circulating half-life. Nowadays, some manufacturers are trying to take advantage of polyethylene glycol (PEG) conjugated with recombinant human growth hormone (rhGH) to improve its half-life and efficacy. However, the modified products are heterogeneous mixtures composed of reaction products with different modification sites. It is generally known as a challenging task to separate and characterize a PEGylated product, especially for its positional isoforms. In this study, cation exchange high performance liquid chromatograph (IEC-HPLC) based on a pH gradient separation method was presented to separate five position isomers of rhGH conjugated with a 40-kDa branched PEG N-hydroxysuccinimidyl (NHS) functional group. Then Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALD-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that each of five materials collected by IEC-HPLC was conjugated with only one branched PEG chain. Furthermore, rhGH and PEG-rhGH were digested by trypsin and peptides were collected by reversed phase high performance liquid chromatography (RP-HPLC). Following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between PEG-rhGH and rhGH. Finally, biological activities of those positional isomers were performed in vivo and very small variations were observed. This method was shown to be suitable for heterogeneity analysis of PEGylated biopharmaceutical products.

A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the Ω-Loop Region.

The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the halflife of the N84S mutant at 50°C increased by 177.89 min when compared with that of the wildtype enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the Tm values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75°C and 80°C, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.

The role of proline substitutions within flexible regions on thermostability of luciferase.

Improving the stability of firefly luciferase has been a critical issue for its wider industrial applications. Studies about hyperthermophile proteins show that flexibility could be an effective indicator to find out weak spots to engineering thermostability of proteins. However, the relationship among flexibility, activity and stability in most of proteins is unclear. Proline is the most rigid residue and can be introduced to rigidify flexible regions to enhance thermostability of proteins. We firstly apply three different methods, molecular dynamics (MD) simulation, B-FITTER and framework rigidity optimized dynamics algorithm (FRODA) to determine the flexible regions of Photinus pyralis luciferase: Fragment 197-207; Fragment 471-481 and Fragment 487-495. Then, introduction of proline is used to rigidify these flexible regions. Two mutants D476P and H489P within most flexible regions are finally designed. In the results, H489P mutant shows improved thermostability while maintaining its catalytic efficiency compared to that of wild type luciferase. Flexibility analysis confirms that the overall rigidity and local rigidity of H489P mutant are greatly strengthened. D476P mutant shows decreased thermosatbility and the reason for this is elucidated at the molecular level. S307P mutation is randomly chosen outside the flexible regions as a control. Thermostability analysis shows that S307P mutation has decreased kinetic stability and enhanced thermodynamic stability.

Complete genome sequence of Acinetobacter baumannii MDR-TJ and insights into its mechanism of antibiotic resistance.

OBJECTIVES: To determine the genome sequence of Acinetobacter baumannii strain MDR-TJ and characterize the mechanisms of multidrug resistance in this strain. METHODS: The whole-genome sequence was determined using Roche 454 GS FLX Titanium. Subsequently, the gaps were closed by sequencing PCR products. The genome of strain MDR-TJ was annotated using IMG ER, the RAST annotation server and the BASys bacterial annotation system. The comM gene of MDR-TJ was examined to identify a possible antibiotic resistance island. Based on the results of multilocus sequence typing, we investigated seven multidrug-resistant A. baumannii strains belonging to global clone 2 (GC2) isolated from Asia, Australia and Europe to determine the backbone shared by resistance islands of GC2 isolates. RESULTS: The A. baumannii strain MDR-TJ genome consists of a circular chromosome and a plasmid, pABTJ1. Strain MDR-TJ was assigned to sequence type ST2. Strain MDR-TJ harbours a 41.6 kb resistance island designated RI(MDR-TJ), which can be derived from the backbone of Tn6167 through the insertion of a Tn6022 into the 3'-end of the tetA(B) gene. Comparative analysis showed that transposon Tn6022 and its truncated forms prevailed in the antibiotic resistance islands of GC2 isolates. The carbapenem resistance gene bla(OXA-23) carried by transposon Tn2009 is located on a putatively conjugative plasmid, pABTJ1. CONCLUSIONS: A. baumannii strain MDR-TJ belongs to GC2 and is resistant to multiple antibiotics. A. baumannii MDR-TJ harbours a genomic resistance island that interrupts the comM gene. The carbapenem resistance of MDR-TJ is mediated by a putatively conjugative plasmid, pABTJ1.

Genome sequence of Acinetobacter baumannii MDR-TJ.

Acinetobacter baumannii is a pathogenic species of bacteria, identified as an aerobic gram-negative bacterium, that is resistant to most antibiotics. In this study, the MDR-TJ strain was isolated at the Second Hospital of Tianjin Medical University, China, and was found to be resistant to penicillin, cephalosporins, aminoglycosides, quinolones, and also imipenem. The genome sequence of Acinetobacter baumannii strain MDR-TJ was determined by using a combination of 454 pyrosequencing and paired-end sequencing performed with the Roche Genome Sequencer FLX system to generate a scaffolded assembly.

Genetic modification of critical enzymes and involved genes in butanol biosynthesis from biomass.

Interest in biobutanol, a sustainable vehicle fuel, is increasing due to rising oil prices and concerns of surrounding climate change and the energy crisis. However, the costs of biobutanol with conventional ABE fermentation by Clostridium are higher than the cost of butanol from today's petrochemical processes. Two major problems in the economic production of biobutanol are difficulty controlling the induction of a metabolic shift from acidogenesis to solventogenesis and limitations imposed by severe product inhibition. With developments in biotechnology, and the completion of genome sequencing of Clostridium, genetic modification is a viable method to improve the solvent yield and the butanol production ratio. The present article aims to highlight the latest research progress on overexpressing, inserting, knocking out, and knocking down genes of the key enzymes in the ABE fermentation pathway and other relative genes (such as genes coding for heat-shock proteins, operon, transcription, etc). Recombinant manipulations of these genes in Escherichiacoli and yeast have also been reported recently, although their butanol yields are lower than in Clostridium. Butanol production with solventogenic clostridia from various feedstocks is also evaluated in this review.

Determination of four water-soluble compounds in Salvia miltiorrhiza Bunge by high-performance liquid chromatography with a coulometric electrode array system.

A method has been developed to determine the four water-soluble components-Danshensu (I), protocatechuic acid (II), protocatechuic aldehyde (III) and salvianolic acid B (IV) in Chinese medicine plant Salvia miltiorrhiza Bunge using high-performance liquid chromatography with a coulometric electrode array detection (HPLC-CEAD) system. Heat reflux extraction was used to pretreat the sample. This analysis was carried on a column of Hypersil C18 (250 mm x 4.6 mm, 5 microm) with a mobile phase of sodium acetate (pH 2.5, 50 mM) and acetonitrile in gradient mode. An ESA electrochemical detector monitored the four compounds. Potentials of four electrodes in series were set at 100, 150, 200 and 250 mV, respectively. Optimization of the pH of mobile phase and the proportion of acetonitrile were also performed. Calibration curve showed good linearity with correlation coefficients (r) more than 0.9937. Average recoveries of the four compounds were more than 92% and relative standard deviations were less than 6.6%. This method appeared to be stable, sensitive and reproducible for determination of the four water-soluble compounds in Chinese medicine plant S. miltiorrhiza Bunge.

Synthesis and activity of an octapeptide inhibitor designed for SARS coronavirus main proteinase.

The outbreak of SARS, a life-threatening disease, has spread over many countries around the world. So far there is no effective drug for the treatment of SARS. Stimulated by the binding mechanism of SARS-CoV Mpro with the octapeptide AVLQSGFR reported recently as well as the "Chou's distorted key" theory, we synthesized the octapeptide AVLQSGFR for conducting various biochemical experiments to investigate the antiviral potential of the octapeptide against SARS coronavirus (BJ-01). The results demonstrate that, compared with other compounds reported so far, AVLQSGFR is the most active in inhibiting replication of the SARS coronavirus, and that no detectable toxicity is observed on Vero cells under the condition of experimental concentration.

Determination of tetracyclines in ovine milk by high-performance liquid chromatography with a coulometric electrode array system.

A method has been developed to analyze residual tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), methacycline (MTC), doxycycline (DC)) in ovine milk, using high-performance liquid chromatography (HPLC) with a coulometric electrode array system. The samples were pretreated, using liquid-liquid extraction based on hexane. The chromatography was performed, using a C18 column (150 mm x 4 mm i.d. and 5 microm) with a mobile phase: sodium phosphate monobasic dihydrate (pH 2.2, 0.05 M)-acetonitrile (78:22, v/v). The flow rate of mobile phase was kept constantly at 1ml/min. The residues were monitored by an ESA electrochemical detector. Potentials of four electrodes in series were set at 400, 660, 680 and 700 mV, respectively. The first electrode was set to remove those interfering substances that may co-elute with TCs and the other three electrodes were used for quantification. The maximal potential of our detection was 700 mV. Calibration curve showed good linearity and the detection limit of TCs was 12.5, 20, 25, 10 and 25 ng/ml, respectively. Optimization of the pH of the mobile phase, the proportion of acetonitrile and the pH of the pretreatment were also performed. Recoveries of TCs from spiked samples were more than 88% and the relative standard deviations were less than 4.3%. This method was reliable, sensitive, economical and suited for routine monitoring of TC residues in ovine dairy milk.

Determination of residual clenbuterol in pork meat and liver by HPLC with electrochemical detection.

AIM: To detect the residual clenbuterol in pork meat and liver using HPLC with Coulometric electrode array system. METHODS: Homogenized meat or liver sample was treated with 1 mol x L(-1) hydrochloric acid and centrifuged, the fat existing in meat or liver tissue was removed by diethyl ether. The pH of the remaining aqueous layer was adjusted to 10.8 +/- 0.2 or 11.6 +/- 0.2 for meat or liver and liquid-liquid extraction with diethyl ether was followed. The ether extract was evaporated to dryness, the residue was dissolved in the mobile phase. The mobile phase A consisted of 50 mmol x L(-1) phosphoric acid-30 mmol x L(-1) triethylamine and was adjusted to pH 4.0 with 2 mol x L(-1) sodium hydroxide solution. The mobile phase B consisted of methanol-acetonitrile (30:45). A mixture of mobile phase A and B (80:20) was used in the method. A four electrode array module was selected for quantitation, the electrode potentials were set at 450, 600, 650 and 680 mV respectively. RESULTS: The two calibration curves for meat and liver showed good linearity between 1.88 - 60.16 ng x g(-1), the detection limit of clenbuterol was 1.2 ng x g(-1). CONCLUSION: This method using HPLC-electrochemical detection is reproducible, and the sensitivity is good enough for the determination of clenbuterol in meat and liver.

Prokaryotic expression of Chinese bovine enterokinase catalytic subunit / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.</p><p><b>METHODS</b>Bovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His.Tag affinity chromatography, and it bioactivity was analyzed.</p><p><b>RESULTS</b>Compared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.</p><p><b>CONCLUSIONS</b>The EKL gene from Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.</p>

[Cloning and fusion expression of bovine enterokinase light chain gene in Escherichia coli].

The objective of the study was to obtain the gene of bovine enterokinase light chain, which would be used in the cleavage and purification of fusion proteins. The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from a sold bovine's duodenal mucosa, then cloned into the pUCmT cloning vector and sequenced. Compared with the sequence deposited in GenBank,the cloned gene sequence is correct. Then the interested gene fragment was inserted into the pET39b expression plasmid. The recombinant vector pET39b-EKL was transformed into E.coli BL21(DE3) and induced by IPTG. It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5'terminal multi-cloning site and recombinant fragment after the analysis of the nucleotide sequence. SDS-PAGE analysis indicated that target product was about 65 kDa which occupied 28% of the total protein. A pure fusion protein was obtained by nickel chelating chromatogram using His*Binding Resin. After desalting and changing buffer, the crude kinase was incubated at 21 degrees overnight and demonstrated a high autocatalytic cleavage activity. This investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.