Surface Mutation Thr34His Facilitates Purification of Haemophilus influenza Carbonic Anhydrase via Metal Affinity Chromatography

In order to pursue Haemophilus influenza carbonic anhydrase (HICA) as a potential drug target, easy and efficient purification methods must be developed. While immobilized metal affinity chromatography (IMAC) may be used, complications with polyhistidine tags is a concern. Inspired by the endogenous metal affinity of Escherichia coli β-carbonic anhydrase (ECCA), we suggest that the generation of histidine clusters on HICA’s surface will facilitate its purification by metal affinity chromatography without the potential interference of His-tags. Here we investigate the Thr34His mutation as a method to generate metal affinity in HICA. Since Thr34His is located only 5.3 Å away from His32, the two residues make a vicinal histidine pair that can interact with nickel resin. We report successful generation of Thr34His HICA mutant plasmid via site-directed mutagenesis. To obtain mutant protein for metal affinity chromatography, Thr34His HICA was overexpressed in E. coli cells and isolated as a cell lysate with a concentration of 20.2 ± 0.6 mg/mL. Metal affinity chromatography was performed on the sample, and the chromatography fractions were analyzed by SDS-PAGE in order to assess the metal affinity of the mutant. SDS-PAGE revealed that while Thr34His HICA eluted at low 10 mM and 25 mM concentrations of imidazole, 150 mM imidazole was required to fully elute the mutant. These results suggest that through the generation of surface histidine pairs, HICA can be engineered to have metal affinity and thus be easily purified via IMAC

DEVELOPMENT OF PAPUA LOCAL WISDOM BASED MATHEMATICS MODULE TO IMPROVE LEARNING OUTCOMES IN GEOMETRY TRANSFORMATION MATERIALS

This research is a development research (Research & Development) which aims to: determine the feasibility of developing a mathematics module based on Papuan local wisdom in class XI students of SMA Negeri 9 Raja Ampat. The development of this module, the researcher refers to the ADDIE development model (Analysis, Design, Development, Implementation, and Evaluation) with an experimental design using a pre test post test one group design, which uses one research class with a sample of 24 students. The module is developed through 5 stages, namely curriculum and needs analysis, module design, module development, testing the module on a small and wide scale and evaluating the module. Material validation results obtained 92.05% and media validation results with 89.18% meaning that the module is suitable for use in learning by revising. Data analysis results using SPSS 26 for a small scale there are 15 valid questionnaire questions from 20 questions, and the average percentage is 90.21, the reliability value is 0.935, this shows that the questionnaire instrument has a very high level of reliability. The large-scale test for students in class XI is 91.41%, meaning that this module is feasible and effective in improving learning outcomes in geometry transformation material. The n-Gain in the four lesson plans is 0.71, which means that the use of mathematics modules based on Papuan local wisdom is very effective in learning