PMID- 32534016
OWN - NLM
STAT- MEDLINE
DCOM- 20210114
LR  - 20210114
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Linking)
VI  - 174
DP  - 2020 Oct
TI  - Modified chemical method for efficient transformation and diagnosis in Pichia 
      pastoris.
PG  - 105685
LID - S1046-5928(20)30145-5 [pii]
LID - 10.1016/j.pep.2020.105685 [doi]
AB  - 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/mug DNA) was observed to 
      be 1.01 x 10(2), 5.07 x 10(3) and 6.52 x 10(3) 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 mug/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.
CI  - Copyright (c) 2020 Elsevier Inc. All rights reserved.
FAU - Kumar, Sandeep
AU  - Kumar S
AD  - Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India; 
      Microbiology & Fermentation Technology Department, CSIR-Central Food 
      Technological Research Institute, Mysore, Karnataka, India.
FAU - Mannil, Aruna
AU  - Mannil A
AD  - PSG College of Arts and Science, Coimbatore, Tamil Nadu, India.
FAU - Mutturi, Sarma
AU  - Mutturi S
AD  - Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India; 
      Microbiology & Fermentation Technology Department, CSIR-Central Food 
      Technological Research Institute, Mysore, Karnataka, India. Electronic address: 
      sarma.mutturi@gmail.com.
LA  - eng
PT  - Journal Article
DEP - 20200610
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - Komagataella pastoris
SB  - IM
MH  - Saccharomycetales/*genetics
MH  - *Transfection
MH  - *Transformation, Genetic
OTO - NOTNLM
OT  - Copy number
OT  - LiCl
OT  - Methods
OT  - Pichia pastoris
OT  - RT-qPCR
OT  - Transformation
EDAT- 2020/06/14 06:00
MHDA- 2021/01/15 06:00
CRDT- 2020/06/14 06:00
PHST- 2020/03/17 00:00 [received]
PHST- 2020/05/01 00:00 [revised]
PHST- 2020/05/30 00:00 [accepted]
PHST- 2020/06/14 06:00 [pubmed]
PHST- 2021/01/15 06:00 [medline]
PHST- 2020/06/14 06:00 [entrez]
AID - S1046-5928(20)30145-5 [pii]
AID - 10.1016/j.pep.2020.105685 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2020 Oct;174:105685. doi: 10.1016/j.pep.2020.105685. Epub 
      2020 Jun 10.