The Study On Tt APuX21 Module

This study is to investigate if Tt APuX21 is a functional carbohydrate-binding module (CBM). The obtained data will help to reveal the binding specificity between Tt APuX21 and chemical bonds. The findings of this study will also help do further study to understand the interaction mechanism between Tt APuX21 and its ligands.Tt apux21 was amplified from Thermoanaerobacter thermohydrosulfuricus by using Ex Taq HS DNA polymerase. The thermocycle parameters were as follows: 1 cycle at 94℃ for 5 minutes, 30 cycles at 94℃ for 30 seconds, 55℃ for 45 seconds and 72℃ for 30 seconds, and 1 cycle at 72℃ for 5 minutes. The PCR product was analyzed by 1% agarose gel electrophoresis. It was apparent that there was an amplified fragment which was about 400bp and was similar in size to the expected one.The PCR product and the expression vector pET21a(+) were similarly digested with restriction endonucleases Nde I and Xho I and were ligated with T4 DNA ligase at 16℃ for 16h. The ligation reaction was then transformed into DH5a.Three single colonies were picked up and cultured in Luria Broth (LB) medium. Bacterial culture PCR was then carried out to identify whether these colonies contained the recombinant plasmid. Agarose gel electrophoresis displayed that all of three showed a specific fragment with the expected size. One of them was sent to be sequenced. Sequencing data showed that it contained the inserted fragment at correct site, and moreover, the sequence of the inserted fragment was the same as that of Tt apux21 published in Genebank (accession number: M28471). The recombinant plasmid was designated as pEX21. The E. coli strain DH5a transformed with pEX21 was grown in LB medium. The recombinant plasmid pEX21 was then extracted and transformed into Tuner.A single colony of E. coli Tuner harboring pEX21 was picked up, inoculated in LB medium and grown to mid-exponential phase (OD₆₀₀=0.6⁰.8). The culture was then cooled to 30℃, before the expression of Tt APuX21 was induced by addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and incubation at 30℃ and 130rpm for a further 5h. Similarly, the E. coli Tuner harboring pET21a(+) was grown and induced as a negative control. Sodium Dodecylsulphate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to analyze the total protein and cell-free extract (CFE) of E. coli Tuner harboring pEX21 and pET21a(+) with and without IPTG induction. The data displayed that there was a wide and bright protein band in lanes containing the total protein and CFE of E. coli Tuner harboring pEX21 with IPTG induction, which was about 20KDa and was similar in size to the deduced molecular weight of Tt APuX21 fusion protein. However, there was no that protein band in other lanes. It indicated that the effective expression of interest protein was obtained.The cells were harvested from 20ml induced culture by centrifugation and lysed by lysozyme. The CFE was then obtained by centrifugation. The fusion protein with a His-tag was purified from the CFE by Metal Ion Affinity Chromatography (MIAC) using Talon? resin (Clontech). The...