Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli

Abstract Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the “one amino acid-one cod...

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Autores principales:	Antti Kulmala, Tuomas Huovinen, Urpo Lamminmäki
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0e24462bc07f43e88130ec2bc2f7bc18
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spelling	oai:doaj.org-article:0e24462bc07f43e88130ec2bc2f7bc182021-12-02T11:41:19ZEffect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli10.1038/s41598-017-03957-62045-2322https://doaj.org/article/0e24462bc07f43e88130ec2bc2f7bc182017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03957-6https://doaj.org/toc/2045-2322Abstract Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the “one amino acid-one codon” method. We redesigned five segments of the Fab gene with a “codon harmonization” method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.Antti KulmalaTuomas HuovinenUrpo LamminmäkiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Antti Kulmala Tuomas Huovinen Urpo Lamminmäki Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
description	Abstract Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the “one amino acid-one codon” method. We redesigned five segments of the Fab gene with a “codon harmonization” method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.
format	article
author	Antti Kulmala Tuomas Huovinen Urpo Lamminmäki
author_facet	Antti Kulmala Tuomas Huovinen Urpo Lamminmäki
author_sort	Antti Kulmala
title	Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
title_short	Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
title_full	Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
title_fullStr	Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
title_full_unstemmed	Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
title_sort	effect of dna sequence of fab fragment on yield characteristics and cell growth of e. coli
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/0e24462bc07f43e88130ec2bc2f7bc18
work_keys_str_mv	AT anttikulmala effectofdnasequenceoffabfragmentonyieldcharacteristicsandcellgrowthofecoli AT tuomashuovinen effectofdnasequenceoffabfragmentonyieldcharacteristicsandcellgrowthofecoli AT urpolamminmaki effectofdnasequenceoffabfragmentonyieldcharacteristicsandcellgrowthofecoli
_version_	1718395384680677376

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli

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