The GenBank accession number of the sample goat LAM gene is JN602369; the plasmid pMD19T-LAM containing the goat lysosomal α-mannosidase gene fragment has been constructed by our laboratory .
The pEASY-Blunt Simple vector is preserved in our laboratory; the yeast expression strain Pichia pastoris GS115 and the Pichia pastoris protein expression vector pPIC9K were presented by Mr. Zhang Yanming, Northwest A&F University.
Goat LAM consists of 949 amino acid residues and five peptides: A (1-298 aa), B (299-382 aa), C (383-541 aa), D (542-823 aa) and E (824- 949 aa). SW and oligosaccharide D-Mannose 5 (D-Mannose 5, abbreviated as man5) were respectively molecularly docked with goat LAM before and after the mutation. According to Kong et al. , AutoDock software was used for molecular docking and mutation analysis of goat LAM genes. In this study, Adobe illustrator CC 2018 software was used to draw a two-dimensional docking map to facilitate observation of the interaction between the two and key amino acids, and groups. The corresponding amino acid position is represented by peptide + colon + amino acid single letter symbol + amino acid sequence number (such as A: H23, which means A-peptide histidine acid residue 23).
Site-directed mutation of goat LAM gene
According to the goat LAM sequence provided in GenBank, Primer Premier 5.0 software was used to design and synthesize primers for wild-type and mutant LAM genes (Table 1). Using the plasmid pMD19T-LAM as the template, LAM F and A: 28 R as the upstream and downstream primers, amplify the DNA fragment containing the 28th tryptophan mutation site; use A: 28 F and D: 599 R as the upper the downstream primers amplify the middle DNA fragment containing two mutation sites; use D: 599 F and LAM R as the upstream and downstream primers to amplify the DNA fragment containing the 599th Tyrosine mutation site. Purify and recover the above DNA fragments, adjust the molar amounts of the three segments to be consistent, and use LAM F and LAM R as upstream and downstream primers to perform overlap extension PCR. Gel electrophoresis detection, recovery of the full-length target fragment of site-directed mutation.
Connect the purified and recovered mutant LAM target gene fragment to the pEASY-Blunt Simple vector (refer to TransGen instructions) to construct a mutant recombinant cloning plasmid, denoted as pEASY-LAMM. With the wild-type recombinant plasmid pEASY-LAM as a control, it was transformed into DH5ɑ competent cells, and positive clones were screened by blue and white spots. Use vector universal primers to screen positive bacteria, extract the positive bacteria plasmids and send them to Shanghai Bioengineering Co., Ltd. for sequencing.
Screening and identification of recombinant Pichia pastoris expression strains
Use His-LAM upstream and downstream primers (see Table 1) to amplify the target gene fragment in pEASY-LAMM. The target gene fragment recovered by EcoR I digestion and purification and the yeast expression vector pPIC9K were ligated overnight at 16°C with T4 ligase to construct the mutant recombinant yeast expression plasmid pPIC9K-LAMM. Transform into E. coli DH5ɑ competent cells, pick a single colony, verify with the primer α-factor F' and the downstream primer His-LAM R of the target fragment, and extract the plasmid for sequencing.
Plasmids pPIC9K-LAM, pPIC9K-LAMM and empty vector pPIC9K were electrotransformed into GS115 yeast competent cells . The electric shock conditions were as follows: voltage 2 kV, time 6 ms. Immediately after the electric shock, 600 μL of ice-cold 1 mol/L sterile sorbitol was added, spread on the RDB plate, and incubated at 30°C for 4-6 d. Pick single colonies and spot them in sequence on yeast extract peptone dextrose medium (YPD) plates containing G418 (concentrations of 50 μg/mL, 100 μg/mL, 150 μg/mL, and 200 μg/mL). Take a single colony grown on the transformation plate with high concentration of G418, and use AOX1 primers to amplify and screen positive bacteria, which are recorded as GS115/pPIC9K-LAM, GS115/pPIC9K-LAMM and GS115/pPIC9K.
Pick a single positive colony and inoculate it into YPD liquid medium, cultivate until OD600 reaches 2-4, and inoculate it in buffered glycerol-complex medium at a ratio of 1%. When OD600 reaches 4-6, centrifuge and add buffer methanol-complex medium to resuspend for bacterial cells, adjust the OD600 of the bacterial solution to about 1.5. Incubate at 30°C and 200 r/min, and add methanol to the culture solution every 24 hours to make the final concentration 1%. Induced and cultured for 96 h, detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Activity determination and characteristic analysis of goat LAM
For the specific enzyme activity determination system and operation, see Kong's report . Each sample was repeated 3 times, and 3 parallel controls were used to calculate the enzyme activity of α-mannosidase based on the absorbance value. The α-mannosidase activity unit (U) is expressed in nanomoles of p-nitrophenol produced by hydrolysis per liter of bacterial liquid per second, that is, nmol∙L-1∙s-1. The sensitivity of the recombinant yeast expression product LAMM to SW is tested by adding 10% SW (m/v) on the basis of the above experiment. The positive control is the recombinant Pichia pastoris expression product wild-type LAM, and the negative control is the Pichia pastoris empty vector expression product. After reacting at 37°C for 1.5 h, the absorbance change was detected under the condition of 405 nm wavelength.
Add equimolar amounts of different metal ions or inhibitors (Ca2+, EDTA, Mn2+, Fe2+, Mg2+, Zn2+, Fe3+, Cu2+, Al3+, Co2+, Cr3+) to the enzyme activity determination reaction system to determine the number of ions with different charges and different species the effect of metal ions on the activity of expressed enzymes; set different reaction temperatures (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C), detect the optimal reaction temperature of LAMM, compare the reaction temperature changes of wild-type and mutant enzymes; prepare different pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) reaction buffer, determine the optimal pH of LAMM, and compare the pH changes of wild-type and mutant enzymes.