Lactococcus lactisas expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins

Abstract

Incorporation of Trp (tryptophan) analogues into a protein may facilitate its structural analysis by spectroscopic techniques. Development of a biological system for the biosynthetic incorpor-ation of such analogues into proteins is of considerable importance. The Gram-negative Escherichia coli is the only prokaryotic expression host regularly used for the incorporation of Trp analogues into recombinant proteins. Here, we present the use of the versatile Gram-positive expression host Lactococcus lactis for the incorporation of Trp analogues. The availability of a tightly regulated expression system for this organism, the potential to secrete modified proteins into the growth medium and the construction of the trp-synthetase deletion strain PA1002 of L. lactis rendered this organism potentially an efficient tool for the incorporation of Trp analogues into recombinant proteins. The Trp analogues 7-azatryptophan, 5-fluorotryptophan and 5-hydroxytryptophan were incorporated with efficiencies of >97, >97 and 89% respectively. Interestingly, 5-methylTrp (5-methyltryptophan) could be incorporated with 92% efficiency. Successful biosynthetical incorporation of 5-methylTrp into recombinant proteins has not been reported previously.

Development of Chemically Defined Media to Express Trp-Analog-Labeled Proteins in a <b><i>Lactococcus lactis</i></b> Trp Auxotroph

“…In this report, new synthetic media have been developed specifically for the production of recombinant alloproteins by the L. lactis Trp auxotroph PA1002, a derivative of L. lactis strain MG1363 [El Khattabi et al, 2008]. Several chemically defined media for L. lactis have been developed over the years [Aller et al, 2014;Jensen and Hammer, 1993;Otto et al, 1983;Poolman and Konings, 1988;Zhang et al, 2009] for metabolic studies, and defined media also allow the expression of heterologous proteins labeled with unnatural amino acids [Berntsson et al, 2009;El Khattabi et al, 2008;Petrovic et al, 2012Petrovic et al, , 2013aPetrovic et al, , 2013bShao et al, 2015].…”

“…The L. lactis Trp auxotroph PA1002 [El Khattabi et al, 2008], which harbors the pMG36e-trprs plasmid for expressing lacTrpRS [Petrovic et al, 2013b], was used in this study. To test the system with two different alloproteins, plasmid pNZ8048-PA295 (containing the gene for the W20 LysM tandem protein [Petrovic et al, 2012]) or plasmid pNSC8048-lmrR (containing the gene for the multidrug transcriptional repressor protein LmrR [Agustiandari et al, 2008]) was electrotransformed into L. lactis PA1002 containing the pMG36e-trprs plasmid.…”

Minimalist IR and fluorescence probes of protein function

“…Broos et al created a tryptophan auxotroph (PA1002) of the gram-positive bacterium Lactococcus lactis [39] that co-expresses L. lactis tryptophanyl-tRNA synthetase (lacTrpRS) under a tightly controlled nisin promoter [40]. This strain is an attractive host for recombinant production of proteins, including membrane proteins.…”

“…This strain is an attractive host for recombinant production of proteins, including membrane proteins. The resulting expression system is the most versatile Trp analog expression system known: it allows for incorporation of Trp analogs such as 7-azatryptophan, 5-hydroxytryptophan, 5-fluorotryptophan and 5-methyltryptophan [39] as well as AzAla [41], the latter cannot be introduced by any other means with high yield and incorporation efficiency.…”

Amino Acid Accumulation Limits the Overexpression of Proteins in Lactococcus lactis

“…A well-tunable nisin-inducible promoter system is available [9], which has been extensively used to produce complex proteins of both pro- and eukaryotic origin [2], [5]–[7]. As a multiple amino acid auxotroph with well-characterized transport systems for amino acids and peptides, L. lactis can readily be employed for incorporating amino acid analogues into proteins, as demonstrated for tryptophan analogues [10] as well as selenomethionine (SeMet) [11]. Especially the latter is of great importance if the protein produced is to be used in crystallographic studies.…”