NDLI: Liquid PTVA: a faster and cheaper alternative for generating multi-copy clones in Pichia pastoris

Content Provider	Springer Nature : BioMed Central
Author	Aw, Rochelle Polizzi, Karen M.
Abstract	Background Multiple cognate gene copy clones have often been used in order to increase the yield of recombinant protein expression in the yeast Pichia pastoris. The method of posttransformational vector amplification (PTVA) has allowed for the efficient generation of multi-copy clones in P. pastoris. However, despite its relative ease and success, this process can be expensive and time consuming. Results We have developed a modified version of PTVA, called Liquid PTVA, which allows for faster and cheaper selection of multi-copy clones. Cultures are grown in liquid medium with only a final selection carried out on agar plates, reducing overall antibiotic usage and increasing the speed of clone amplification. In addition, it was established that starting PTVA with a single copy clone resulted in higher copy number strains for both traditional plate PTVA and liquid PTVA. Furthermore, using the Zeocin selection marker in liquid PTVA results in strains with higher growth rates, which could be beneficial for recombinant protein production processes. Conclusions We present a methodology for creating multi-copy clones that can be achieved over 12 days instead of the traditional 45 and at approximately half the cost.
Related Links	https://microbialcellfactories.biomedcentral.com/counter/pdf/10.1186/s12934-016-0432-8.pdf
Ending Page	11
Page Count	11
Starting Page	1
File Format	HTM / HTML
ISSN	14752859
DOI	10.1186/s12934-016-0432-8
Journal	Microbial Cell Factories
Issue Number	1
Volume Number	15
Language	English
Publisher	BioMed Central
Publisher Date	2016-02-05
Access Restriction	Open
Subject Keyword	Applied Microbiology Biotechnology Microbiology Microbial Genetics and Genomics Enzymology Genetic Engineering Pichia pastoris/Komagataella phaffii Multi-copy clones Posttransformational vector amplification Recombinant protein expression
Content Type	Text
Resource Type	Article
Subject	Applied Microbiology and Biotechnology Bioengineering Biotechnology
Journal Impact Factor	4.3/2023
5-Year Journal Impact Factor	5.5/2023

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