Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture

Aims@#Hyaluronic acid (HA) is a high molecular weight polymer and a major component of mucoid capsule in bacteria and extracellular matrix (ECM) of vertebrate tissue. Due to its unique characteristics, HA is used extensively in medical and cosmetic field. However, because of the exotoxins production from animal tissues extraction and Streptococcus zooepidemicus, HA production by recombinant microorganisms has gained interest. The present study was aimed at cloning of hasA gene in Escherichia coli and optimization of the medium components for HA production. @*Methodology and results@#A fragment of an approximate size of 1.5 kb that encodes the hyaluronan synthase (hasA) gene from S. zooepidemicus ATCC 39920 was amplified by PCR using hasA-specific primers. The hasA gene was ligated into the bacterial expression vector pLbADH and transformed into the expression host, Escherichia coli BL21 strain. Then, genetically engineered E. coli strain BL21 was used for the production of HA by fermentation using different glucose concentration (10-50 g/L) and different IPTG concentration (0.1, 0.5 and 1.0 mM) in shake flask culture. Amongst varying glucose concentrations, results showed that 50 g/L glucose with nutrient rich media containing nitrogen source was able to produce the highest HA concentration (0.115 ± 0.002 g/L). With addition of 1.0 mM IPTG, HA production reached a peak 0.532 ± 0.026 g/L which is around fivefold higher compared to without IPTG. @*Conclusion, significance and impact of study@#The hasA gene was cloned from S. zooepidemicus and successfully expressed in recombinant E. coli BL21 cells. This low molecular weight HA is gaining more importance in medical and cosmetic application due to possess pronounced free radical scavenging and antioxidant activities.

A new protecting method for the inferior parathyroid gland in situ: the meticulous thyrothymic ligament dissection technique / 中华内分泌外科杂志

Objective To explore the anatomical features and the dissection technique of thyrothymic ligament (TTL),and to explore the clinical significance of protecting the inferior parathyroid gland (IPTG) with this structure.Methods Patients who received the initial thyroid surgery in the Department of Thyroid Surgery of the First Affiliated Hospital with Nanjing Medical University from May.2017 to Dec.2017 were prospectively analyzed.We dissected TTL,identified and located the IPTG,described the structural features of TTL,and investigated the position relationship of TTL and IPTG to evaluate the possibility and value of protecting IPTG in situ.Results About 121 patients underwent the dissection,totally 194 sides dissected that included 96 left sides and 98 right sides.TTL was found in 143 sides (73.7％),78 left sides (81.3％) and 65 right sides (66.3％).Nearly 70.6％ IPTG can be proactively identify and located by the TTL during the operation.TTL was a kind of adipose connective tissue that was wide at the bottom and narrow at the top,accompanying with the inferior thyroid vein,from the thymus to the thyroid.76.2％ TTL were attached to the lower pole and the lower 1/3 dorsal of thyroid,containing fat and vessels.33.5％ IPTGs were located in the area surrounding around the ends of the TTL.25.3％ IPTGs were located in the TTL.4.6％ IPTGs were located in the thymus and 7.2％ IPTGs surrounding around the TTL.The incidence rate of post-operation hypoparathyroidism was 14.9％.Conclusions TTL commonly exists and has significant relationship with IPTG.TTL connects thymus and IPTG,which would be considered a complex (thymus-thyrothymic ligament-IPTG complex,TLIC).The meticulous TTL dissection technique will help proactively identify,locate and protect IPTG during operation,and reduce the incidence rate of post-operation hypoparathyroidism.

Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

EVALUATION OF THE FEASIBILITY OF USING LACTOSE AS THE INDUCER IN PRODUCING THE VIRAL ENHANCING FACTOR FROM HELICOVERPA ARMIGERA GRANULOSIS VIRUS IN ESCHERICHIA COLI BL21DE3 / 微生物学通报

Lactose was shown to no less competent than Isopropyl-?-D-thiogalactoside (IPTG) in inducing the expression of the ENHANCIN coding gene from Helicoverpa armigera granulosis virus in Eswcherichia coli BL21 (DE3) regulated by a T7 promoter, since the lactose induction could lead to an ENHANCIN band no smaller than the one in IPTG induction on the SDS-PAGE gel. This would decrease the cost of the large-scale ENHANCIN production. The lactose concentration was optimized at 2.2% - 2.5% (w/v) . Different treatments on the lactose sterilization showed that lactose steam- sterilized in 116. 5℃ for 15min could lead to the ENHANCIN production. The convenience and the relatively low cost in its" operation could further decrease the cost of the ENHANCIN production.