Recombinant wheat ALMT1 protein expression in Pichia pastoris

Author: Mona Kaspal

Kaspal, Mona, 2021 Recombinant wheat ALMT1 protein expression in Pichia pastoris, Flinders University, College of Medicine and Public Health

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Gamma aminobutyric acid (GABA), is a non-protein amino acid, found in all kingdoms of life (archaea, bacteria and eukaryotes) (Bouché, Lacombe, et al. 2003). In mammalian brains, binding of GABA to the GABAA receptor activates chloride channels allowing chloride influx to the cells and regulates neuronal firing during stress (Palacios et al. 1981). In plants, GABA has been shown to be a metabolite that changed under abiotic and biotic stresses (MacGregor et al. 2003; Mekonnen et al. 2016; Perez-Alfocea et al. 1994; Ramputh & Bown 1996; Rhodes et al. 1986). However, recent discovery of a putative GABA binding site on a family of anion channels (Aluminium Activate Malate Transporters – ALMT) involved in aluminium stress tolerance suggests that GABA regulates these channels and may act as a stress signalling molecule (Ramesh et al. 2015).

The discovery of a putative binding site (12 amino acid stretch) on the wheat ALMT (TaALMT1) as a transducer of GABA signalling has broadened the area of research into the plant signalling pathways (Ramesh et al. 2015). However, the structure or topology of TaALMT1 or other ALMTs is not known and the molecular determinants of GABA regulation of anion channels is not yet completely understood (Fromm 2020). Thus, it is difficult to identify the exact physiological role of GABA and its role in stress signalling and tolerance in plants.

In this project TaALMT1 was expressed in the yeast Pichia pastoris with the aim of purifying the recombinant protein for determining its tertiary or 3D structure via Cryo-EM studies and bioinformatics was used to identify other plant proteins with putative GABA binding sites to understand the GABA regulation.

Amplified ALMT1 was cloned into pPICZB with XhoI and XbaI restriction sites followed by transformation into E. coli competent cell DH5α. Plasmid DNA was extracted, sequenced to confirm the DNA sequence, and then transformed into Pichia pastoris. In the process of colony PCR to confirm integration, Thermopol Taq polymerase (NEB) was found to be more suitable than Go Taq Polymerase (Promega) and X33 strain of Pichia was better suited for expression of protein than SMD1168 strain. 24-48 hours of induction with methanol was sufficient for protein induction. Following transformation into Pichia, protein was induced by addition of methanol over a period of 24-120 h. The cells of the Pichia were disrupted using glass beads in a bead beater and induction of protein expression confirmed using anti-His antibody via dot blot. However, protein expression could not be confirmed via western blots. Future work is to optimise protein induction followed by affinity purification of the recombinant protein for tertiary structure determination.

Further to identify the other proteins that might have a putative GABA binding site, a consensus sequence DVFXXXXWXXEXL, based on different plant ALMTs and mammalian GABA receptors was used in a BLAST search. The search identified plant ALMTs as well other proteins. However, 20 different proteins from both monocot and dicot plants were with the putative GABA binding sites selected for this study. highly conserved amino acids Valine, Phenylalanine and Tryptophan which have been shown to be important for GABA regulation. These proteins need to be functionally characterized in the future to determine if they are regulated by GABA.

Keywords: Biotic and Abiotic stresses, GABA, ALMT 1, Pichia pastoris, protein expression

Subject: Biological Sciences thesis

Thesis type: Masters
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Dr. Sunita Ramesh