Conjugation of α-amylase with dextran for enhanced stability: Process details, kinetics and structural analysis

The influence of enzyme polysaccharide interaction on enzyme stability and activity was elucidated by covalently binding dextran to a model enzyme, α-amylase. The conjugation process was optimized with respect to concentration of oxidizing agent, pH of enzyme solution, ratio of dextran to enzyme concentration, temperature and time of conjugate formation, and was found to affect the stability of α-amylase. α-Amylase conjugated under optimized conditions showed 5% loss of activity but with enhanced thermal and pH stability. Lower inactivation rate constant of conjugated α-amylase within the temperature range of 60-80°C implied its better stability. Activation energy for denaturation of α-amylase increased by 8.81kJ/mol on conjugation with dextran. Analysis of secondary structure of α-amylase after covalent binding with dextran showed helix to turn conversion without loss of functional properties of α-amylase. Covalent bonding was found to be mandatory for the formation of conjugate.     

Expression, purification and development of neutralizing antibodies from synthetic BoNT/B LC and its application in detection of botulinum toxin serotype B

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) neurotoxins (BoNTs). The mouse bioassay is the gold standard for the detection of botulinum neurotoxins, however it requires at least 3-4 days for completion. Most of the studies were carried out in botulinum toxin A and less on type B. Attempts have been made to develop an ELISA based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. In the present study, the synthetic BoNT/B LC gene was constructed using PCR overlapping primers, cloned in a pET28a+ vector and expressed in E. coli BL21DE3. The maximum yield of recombinant proteins was optimized after 16 hrs of post induction at 21°C and purified the recombinant protein in soluble form. Antibodies were raised in Mice and Rabbit. The IgG antibody titer in the case of Mice was 1: 1,024,000 and Rabbit was 1: 512,000 with alum as adjuvant via intramascular route. The biological activity of the recombinant protein was confirmed by in-vitro studies using PC12 cells by the synaptobrevin cleavage, the rBoNT/B LC protein showed the maximum blockage of acetylcholine release at a concentration of 150nM rBoNT/B LC in comparison to the control cells. When the cells were incubated with rBoNT/B LC neutralized by the antisera raised against it, the acetylcholine release was equivalent to the control. IgG specific to rBoNT/B LC was purified from raised antibodies. The results showed that the developed antibody against rBoNT/B LC protein were able to detect botulinum toxin type B approximately up to 1 ng/ml. These developed high titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.     

Controlled release of nutrients to mammalian cells cultured in shake flasks

Though cell culture-based protein production processes are rarely carried out under batch mode of operation, cell line and initial process development operations are usually carried out in batch mode due to simplicity of operation in widely used scale down platforms like shake flasks. Nutrient feeding, if performed, is achieved by bolus addition of concentrated feed solution at different intervals, which leads to large transient increases in nutrient concentrations. One negative consequence is increased waste metabolite production. We have developed a hydrogel-based nutrient delivery system for continuous feeding of nutrients in scale down models like shake flasks without the need for manual feed additions or any additional infrastructure. Continuous delivery also enables maintaining nutrient concentrations at low levels, if desired. The authors demonstrate the use of these systems for continuous feeding of glucose and protein hydrolysate to a suspension Chinese Hamster Ovary (CHO) culture in a shake flask. Glucose feeding achieved using the glucose-loaded hydrogel resulted in a 23% higher integral viable cell density and an 89% lower lactate concentration at the end of the culture when compared with a bolus-feed of glucose.     

Mining and gene ontology based annotation of SSR markers from expressed sequence tags of Humulus lupulus

Humulus lupulus is commonly known as hops, a member of the family moraceae. Currently many projects are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. The genetically characterized domains in these databases are limited due to non-availability of reliable molecular markers. The large data of EST sequences are available in hops. The simple sequence repeat markers extracted from EST data are used as molecular markers for genetic characterization, in the present study. 25,495 EST sequences were examined and assembled to get full-length sequences. Maximum frequency distribution was shown by mononucleotide SSR motifs i.e. 60.44% in contig and 62.16% in singleton where as minimum frequency are observed for hexanucleotide SSR in contig (0.09%) and pentanucleotide SSR in singletons (0.12%). Maximum trinucleotide motifs code for Glutamic acid (GAA) while AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking primer pairs were designed in-silico for the SSR containing sequences. Functional categorization of SSRs containing sequences was done through gene ontology terms like biological process, cellular component and molecular function.     

Chaperone Hsp27, a novel subunit of AUF1 protein complexes, functions in AU-rich element-mediated mRNA decay

Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3' untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1- and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (ARE-mRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.     

Evidence of Native-like Substructure(s) in Polypeptide Chains of Carbonic Anhydrase Deposited into Insoluble Aggregates During Thermal Unfolding

A non-specific protease, subtilisin, was used to probe the existence of folded structure in thermal aggregates of bovine carbonic anhydrase-II (BCA). BCA aggregates and native BCA were subjected to proteolysis and electrophoretic analyses which revealed the accumulation of polypeptide fragments of similar size, indicating survival of similar sections of folded structure burying peptide bonds away from scission in the two samples. N-terminal sequencing revealed that the termini of size-matched fragments from the two samples were either identical, or located very close to each other, and predominantly on the surface of the 3-dimensional structure of native BCA. The susceptibility to proteolysis of very nearly the same sites in the two samples suggests that native-like elements of structure survive within BCA aggregates. The finding that thermal aggregation can involve interactions among molecules retaining elements of native-like structure, suggests that complete chain unfolding may not be a necessary prerequisite for all aggregation.     

Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth

Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth.