M T Hossain*
Department of Biochemistry and Molecular Biology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Y Aso
Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
sHSPs, Pre-incubation, CLA, BLC, Silkworm, sHSP19.9, sHSP20.8
Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Animal Health and Management
Materials Unless otherwise noted, the buffers used were 50 mM sodium phosphate buffer (pH 8.0) containing 0.1 M NaCl (buffer A), containing 0.3 M NaCl (buffer B), and 20 mM HEPES buffer (pH 7.7) containing 10 mM NaCl (buffer C). DTT was used as 10 mM. BLC was prepared with Milli Q (2 mm) water without further preparation. All of the used chemicals were reagent grade and freshly prepared. The incubation times were selected as 15, 20, 45, 60 and 240 minutes based on the requirement. Purification of sHSP19.9 and sHSP20.8 sHSP20.8 and sHSP19.9 were prepared as recombinant N-terminal His-tagged briefly described below. Pre-culture and culture of cells Each of sHSP19.9 and sHSP20.8 genes was sub-cloned into a pET28a expression vector. The resulting plasmids were introduced into the (DE3) E. coli cells and cultured at 37ºC for 12 to 15 hour. Thus sHSP19.9 and sHSP20.8 were overproduced as recombinant proteins having N-terminal His-tags. The entire procedure of purification was followed as described by Sakano et al. (2006) and Aso et al. (2007) with minor modifications (Hossain et al., 2010). The cells were grown up at 37ºC on Luria-Bertani (LB) medium (Sigma, USA) until mid-log phase where absorbance at 600 nm was within 0.5 to 0.6. After induction with 1 mM Isopropyl β-D-1thiogalactopyranoside (IPTG) (Wako Pure Chemical Industries Ltd. Tokyo, Japan), the cells were further cultured for 3 hour and collected from 150 ml of LB medium by centrifugation at 1,095 × g for 20 minutes. The resulting cell pellet was made frozen with liquid nitrogen and stored at -70ºC until use. Isolation and purification of sHSPs from cells The pellet was suspended in 30 ml of buffer A and centrifuged at 1,095 × g for 20 minutes at 4ºC. The resultant pellet was re-suspended with 10 ml of the same buffer and was lysed by sonication for 1 hour. Insoluble substance was removed by addition of 0.33 ml of 5% polyethyleneimine (MP Biomedical Inc., Germany) and 1 ml of 5.0 M NaCl and was stirred on ice for 1 hour and centrifuged at 17,528 × g for 20 min. The resulting supernatant was taken, and soluble proteins were precipitated with 80% saturation of ammonium sulfate by stirring on ice for more than 1 hour. After centrifugation at 7,243 × g for 20 min, the resulting precipitate was suspended with buffer C, kept at 4ºC for 1 hour, and centrifuged at 7,243 × g for 20 minutes. The resulting supernatant was saturated with 80% of ammonium sulfate to precipitate proteins. It was done on ice for more than 1 hour and centrifuged at 7,243 × g for 20 minutes. The resulting pellet was suspended with 10 ml buffer B containing 45 mM imidazole and poured into the suspension of Ni-NTA resin pre-equilibrated with the same buffer, and churned for 1 hour at 4ºC. The resin was washed with buffer B containing 45 mM imidazole, and sHSPs were thus eluted with the buffer B containing 250 mM imidazole. Four fractions (1 ml each) were collected. Based on the higher absorbance at 280 nm of the eluted proteins, two fractions were mixed together and dialyzed overnight against buffer C at 4ºC. sHSP20.8 against BLC aggregation sHSP20.8 with buffer C was used against BLC aggregation in two conditions: with preincubation and without pre-incubation. Both time- and concentration-dependent CLA of sHSP20.8 were monitored by Shimadzu UV2400 double beam spectrophotometer at 360 nm wavelength. For the former case, 2.76 µM sHSP20.8 was used against 8.96 µM BLC and the activities were continuously monitored from 0 to 50 minutes. For the later one, different molar ratios; 1:0 (BLC 5.0 µM), 1:0.2, 1:0.5, 1:1 and 1:2 were used in the cell and the performance was observed at 360 nm. The interaction between incubated BLC (at 60ºC) and sHSP20.8 at 60ºC also was observed prior to investigate the CLA of pre-incubated sHSP20.8. BLC (14.0 µM) was incubated for 5 and 7.5 minutes in the cell prior to addition of sHSP20.8 (7.0 µM). The absorbance was monitored for 15 minutes. The entire process was repeated again at 30ºC with the same conditions and methods. Moreover, BLC (0.74 µM) was incubated at 60ºC for 2, 4, 6, 8, 10, 20 and 40 minutes and centrifuged at 10,955 × g for 5 minutes. The resultant supernatant was used again to monitor the interaction with 7.4 µM sHSP20.8. Effects of pre-incubated sHSP20.8 on the CLA were monitored then. sHSP20.8 (2.5 µM) with buffer C was incubated for different times; 5, 10, 20 and 30 minutes prior to apply against BLC (5.0 µM) aggregation with control (without pre-incubation). The used molar ratios for both cases were fixed at 1:0.5 (BLC and sHS20.8). Concentration-dependent pre-incubation effects of sHSP20.8 were also observed using different molar ratios; 1:0 (control), 1:0.01, 1:0.05 and 1:0.1. sHSP20.8 and buffer C (control). They were incubated for 30 min in the cell and fixed amount of BLC (7.0 µM) was added to it. Keeping the molar ratio fixed at 1:1 but using higher (40.0 µM) and lower concentration (5.0 µM) of each sHSP20.8 and BLC, the CLA of 30 minutes pre-incubated sHSP20.8 was further monitored for 4 hours. sHSP19.9 against BLC aggregation By using the similar method as mentioned above, 30 minutes pre-incubated sHSP19.9 with 10 mM DTT were used to observe the preincubation effects. The respective DTT containing sHSP19.9 with buffer C was incubated for 30 minutes and fixed amount of BLC (5.0 µM) was added to it. The absorbance was taken at 360 nm. Different molar ratios such as 1:0.25, 1:0.5 and 1:0.75 for BLC to sHSP19.9 were used to observe the effects of pre-incubation on CLA of sHSP19.9 , whereas the same ratios were used as control (without pre-incubated). Data analysis and interpretation The used data were analysed and figures were prepared by using OriginPro7.5 Scientific Graphing and Analyzing Software (Origin Lab).
Bang. J. Anim. Sci. 2017. 46 (4):258-265
Journal