Modified chemical method for efficient transformation and diagnosis in Pichia pastoris.

Sandeep, Kumar and Aruna, M. and Sarma, Mutturi (2020) Modified chemical method for efficient transformation and diagnosis in Pichia pastoris. Protein Expression and Purification, 174. p. 105685. ISSN 1046-5928

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Abstract

In the present study, green fluorescence protein (GFP) was used as a candidate protein to test and optimize a robust chemical transformation procedure in P. pastoris. Towards this, it was adjudged that pretreatment of P. pastoris cells with lithium chloride (LiCl) and its optimal concentration is critical for efficient transformation. Using three different methods (M1: 100 mM LiCl, 10 min, M2: 1 M LiCl, 10 min and M3: 1 M LiCl, 1 h), it was found that concentration and incubation time for LiCl treatment significantly affects the transformation efficiency. The transformation efficiency (transformants/μg DNA) was observed to be 1.01 × 102, 5.07 × 103 and 6.52 × 103 using methods M1, M2 and M3, respectively, indicating the superiority of M3. Moreover, presence of the GFP gene in the positive transformants was confirmed using a novel colony PCR method where the colonies were treated with LiCl prior to GFP specific amplification. Also, it was established using fluorescence microscopy and western blot analysis that increasing zeocin concentration as a post transformational vector amplification (PTVA) strategy increased the fluorescence and gene expression, respectively. Further, RT-qPCR revealed that the gene copy number using methods M1, M2 and M3 were 2.97, 5.26 and 7.19, respectively, when 500 μg/ml zecocin was used for selection, thus corroborating western blot results. In conclusion, we demonstrate a cheap and robust chemical method for achieving higher transformation efficiency in P. pastoris and a simple procedure for colony-PCR based-diagnosis alleviating the need for enzymatic treatment.

Item Type:	Article
Uncontrolled Keywords:	Pichia pastoris,LiCl Transformation, Copy number RT-qPCR Methods
Subjects:	600 Technology > 08 Food technology > 16 Nutritive value > 03 Proteins
Divisions:	Fermentation Technology and Bioengineering
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	06 Oct 2020 09:32
Last Modified:	06 Oct 2020 09:32
URI:	http://ir.cftri.res.in/id/eprint/14514

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