scholarly journals Development of Chemically Defined Media to Express Trp-Analog-Labeled Proteins in a Lactococcus lactis Trp Auxotroph

2016 ◽  
Vol 26 (4) ◽  
pp. 269-276
Author(s):  
Jinfeng Shao ◽  
Marcelo F.M. Marcondes ◽  
Vitor Oliveira ◽  
Jaap Broos
Keyword(s):  
Amino Acids ◽  
New Media ◽  
Lactococcus Lactis ◽  
Unnatural Amino Acids ◽  
Defined Medium ◽  
Trna Synthetase ◽  
Growth Media ◽  
Heterologous Proteins ◽  
Chemically Defined Media ◽  
Defined Media

Chemically defined media for growth of <i>Lactococcus lactis</i> strains contain about 50 components, making them laborious and expensive growth media. However, they are crucial for metabolism studies as well as for expression of heterologous proteins labeled with unnatural amino acids. In particular, the <i>L. lactis</i> Trp auxotroph PA1002, overexpressing the tryptophanyl tRNA synthetase enzyme of <i>L. lactis</i>, is very suitable for the biosynthetic incorporation of Trp analogs in proteins because of its most relaxed substrate specificity reported towards Trp analogs. Here we present two much simpler defined media for <i>L. lactis</i>, which consist of only 24 or 31 components, respectively, and with which the <i>L. lactis</i> Trp auxotroph shows similar growth characteristics as with a 50-component chemically defined medium. Importantly, the expression levels of two recombinant proteins used for evaluation were up to 2-3 times higher in these new media than in the 50-component medium, without affecting the Trp analog incorporation efficiency. Taken together, the simplest chemically defined media reported so far for <i>L. lactis</i> are presented. Since<i> L. lactis</i> also shows auxotrophy for Arg, His, Ile, Leu Val, and Met, our simplified media may also be useful for the biosynthetic incorporation of analogs of these five amino acids.

scholarly journals Effect of methionine and cysteine deprivation on growth of different natural isolates of Lactobacillus spp. in chemically defined media

2008 ◽  
Vol 60 (4) ◽  
pp. 509-517 ◽  
Author(s):  
Jelena Lozo ◽  
Jelena Begovic ◽  
B. Jovcic ◽  
Natasa Golic ◽  
L. Topisirovic
Keyword(s):  
Amino Acids ◽  
Defined Medium ◽  
Ecological Niches ◽  
Chemically Defined Medium ◽  
Chemically Defined Media ◽  
Defined Media ◽  
Natural Isolates ◽  
Lactobacillus Spp

The purpose of this study was to determine the ability of natural isolates of lactobacilli from different ecological niches to grow in a chemically defined medium in the presence or absence of sulphur-containing amino acids, methionine and/or cysteine. The obtained results indicate that cysteine is essential for growth of L. paracasei subsp. paracasei BGHN14 and BGSJ2-8, while methionine is essential for isolates BGHN40, BGCG31, and BGHV54T of the species L. plantarum. Methionine is also essential for growth of L. rhamnosus BGHV58T. Other analyzed strains, such as L. plantarum BGSJ3-18, BGZB19, BGHV52Ta, and BGHV43T, require the presence of both amino acids for their growth.

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

scholarly journals Expanding the Library and Substrate Diversity of the Pyrrolysyl-tRNA Synthetase to Incorporate Unnatural Amino Acids Containing Conjugated Rings

ChemBioChem ◽  
2013 ◽  
Vol 14 (16) ◽  
pp. 2100-2105 ◽  
Author(s):  
Vanessa K. Lacey ◽  
Gordon V. Louie ◽  
Joseph P. Noel ◽  
Lei Wang
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase

Cultivation and properties of Neisseria sp. grown in chemically defined media

1972 ◽  
Vol 18 (7) ◽  
pp. 1087-1090 ◽  
Author(s):  
C. P. Kenny ◽  
B. B. Diena ◽  
R. Wallace ◽  
L. Greenberg
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Present Report ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Chemically Defined Media ◽  
Defined Media ◽  
Specific Antisera

Neisseria Chemically Defined Medium (NCDM) has been used routinely in our laboratory for a variety of purposes. The present report describes the development of NCDM agar, wherein the NCDM base is sterilized by filtration and defined supplements and agar are added. The medium is transparent and both meningococci and gonococci grow within 72 h. When grown on NCDM agar, Types 2 and 3 gonococcal colonies tend to revert to Type 1. The serological grouping of meningococci with specific antisera is not affected by growth on this medium.Parallel investigations on the growth of these species in liquid NCDM demonstrated that the yield of Neisseria gonorrhoeae is enhanced when the medium is sterilized by filtration.

scholarly journals Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Biomolecules ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 255 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yaroslav Andreev
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Nonsense Suppression ◽  
Expression Vectors ◽  
Site Specific ◽  
Suppressor Trna ◽  
E Coli ◽  
Orthogonal Pair

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

scholarly journals Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair

2010 ◽  
Vol 132 (42) ◽  
pp. 14819-14824 ◽  
Author(s):  
Susan M. Hancock ◽  
Rajendra Uprety ◽  
Alexander Deiters ◽  
Jason W. Chin
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase

Chemically defined media and auxotrophy of the prolific l-lactic acid producer Lactococcus lactis IO-1

2013 ◽  
Vol 115 (5) ◽  
pp. 481-484 ◽  
Author(s):  
Miki Machii ◽  
Satoru Watanabe ◽  
Takeshi Zendo ◽  
Taku Chibazakura ◽  
Kenji Sonomoto ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Chemically Defined Media ◽  
Defined Media
Sign in / Sign up

Export Citation Format

Share Document