Development of an Efficient Method for Generating and Screening Active Trypsin and Trypsin Variants

Evnin, Luke B.; Craik, Charles S.

doi:10.1111/j.1749-6632.1988.tb25808.x

Cited by 29 publications

(25 citation statements)

References 35 publications

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“…Escherichia coli has been the host of choice for the production of rat trypsin, and altered forms of rat trypsin have been reported to be produced at levels of as high as 1 mg/liter (15). More recently, rat trypsinogen was produced in Saccharomyces cerevisiae with yields of up to 10 mg/liter (20,38).…”

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A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

Hanquier

Sorlet

Desplancq

et al. 2003

Appl Environ Microbiol

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We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation.

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confidence: 99%

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

Hanquier

Sorlet

Desplancq

et al. 2003

Appl Environ Microbiol

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“…Mutagenesis of pLIB with a pool of oligonucleotides comprising all possible nucleotide combinations at the positions encoding amino acids 189 and 190 (5'-CTAGAGGGAGGCAAGNNNNNNTGCCAGGGC-GACTCTGGTGGGCC-3'; N = A, C, G, or T) was performed as described (22) except that uracil-laden single-stranded DNA was isolated from strain LE112. It was anticipated that each of the oligonucleotides in the pool would anneal with roughly equal efficiency to the pLIB template since the oligonucleotides had an equal number of bases complementary to the template.…”

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confidence: 99%

“…Trypsin was harvested from 1.5 ml of an E. coli X90 culture grown to saturation in LB/ampicillin (50 mg/ liter)/1 mM IPTG (22). For the microplate assay, 20 jul of the 40 p.l of extract was added to 180 jul of 100 mM Tris*HCl, pH 8.0/0.4 mM t-Boc-Leu-Gly-Arg p-nitroanilide in 1 well of a 96-well microplate.…”

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confidence: 99%

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Substrate specificity of trypsin investigated by using a genetic selection.

Evnin

Vásquez²,

Craik³

1990

Proc. Natl. Acad. Sci. U.S.A.

147

110

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The structural determinants of the primary substrate specificity of rat anionic trypsin were examined by using oligonucleotide-directed mutagenesis coupled to a genetic selection. A library was created that encoded trypsins substi- Trypsin is a paradigm for the family of serine proteases that have evolved to cleave peptide bonds after Arg and Lys amino acid residues (9-11). The substrate specificity exhibited by this enzyme family toward these two structurally disparate, positively charged amino acids is defined by the topography of the enzyme, particularly the primary specificity pocket. Crystallographic analysis of trypsin-inhibitor complexes (12-14) and mutagenesis of amino acids that comprise the substrate binding pocket (1,15,16) indicated that two positions, 189 and 190 (trypsin amino acid numbering is based on the chymotrypsinogen amino acid numbering described in ref. 17), are critical in defining the substrate specificity ofthe enzyme. It was anticipated that substitution at these positions would alter function, with a minimum disruption of the structure of the binding pocket, because the remainder of the interactions between the enzyme and the substrate side chain are mediated by main-chain atoms (1).We wished to examine the functional contribution to Arg and Lys specificity of the two amino acids at the base of the substrate binding pocket. A library encoding trypsins substituted at positions 189 and 190 was constructed, and a genetic selection was developed to test the activity of these proteins. A set of mutant trypsins with partially preserved function was isolated and kinetically characterized to investigate the components of Arg and Lys specificity.

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“…The resulting plasmid, pVNA14, was transformed into E. coli CJ236 for synthesis of uracil-laden, single-stranded DNA (31). The PnifA FIXK binding site (anaerobox), NIFA binding site (NIFAbox), and 54 binding site ( 54 box) regions were mutagenized in all possible combinations as described previously (20) with the following synthetic oligonucleotides:…”

Section: Methodsmentioning

confidence: 99%

Interactive regulation of Azorhizobium nifA transcription via overlapping promoters

1995

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The Azorhizobium nifA promoter (PnifA) is positively regulated by two physiological signal transduction pathways, NtrBC, which signals anabolic N status, and FixLJK, which signals prevailing O 2 status. Yet, PnifA response (gene product per unit time) to these two activating signals together is more than twice that of the summed, individual signals. In the absence of NIFA, a negative PnifA autoregulator, the fully induced PnifA response is more than 10-fold greater than that of summed, individual signals. Given this synergism, these two signal transduction pathways must interactively regulate PnifA activity. Unique among characterized bacteria, Azorhizobium caulinodans fixes N 2 at high rates both in free-living culture and in symbiosis with Sesbania spp. host legumes (17). Both structural and regulatory (nif) genes for N 2 fixation are strongly conserved across the Bacteria. Among the latter is the nifA gene, which encodes a master transcriptional activator. While nifA coding sequences are highly conserved, nifA expression patterns are quite different, even among related N 2 -fixing bacteria. Different nifA regulatory circuits reflect orthologous cisand trans-acting elements ''wired'' together quite differently (21). Klebsiella pneumoniae, a microaerophilic diazotroph, regulates nifA expression hierarchically through two signal transduction pathways. The primary pathway, low anabolic N signal 3 NTRB 3 NTRC 3 nifLA, allows NIFA synthesis, while a secondary pathway, high O 2 signal 3 NIFL Oٜ NIFA 3 nifLA, modulates NIFA levels (Oٜ, negative regulation; 3, positive regulation). In K. pneumoniae, both NTRB3NTRC and NIFL Oٜ NIFA constitute cognate pairs of environmental sensor and response regulator. The NTRB3NTRC pair, when stimulated by anabolic N limitation, activates transcription at specific targets, among them the nifLA operon (18). In an amplification cascade, K. pneumoniae NIFA so produced not only activates transcription at other nif operons, it also autoregulates its own transcription (7). NIFA negatively responds to its cognate environmental sensor, NIFL, which somehow monitors endogenous O 2 , and to high anabolic N levels (26,36). Both NIFA and NTRC specifically bind DNA and activate RNA polymerase 54 -type transcription initiation complexes. In K. pneumoniae, promoter targets carry distinct upstream activating elements which bind either NIFA or NTRC (8,47) as well as diagnostic Ϫ12 and Ϫ24 elements, which bind 54 initiator either alone or complexed with core RNA polymerase (5, 6). This integration of cis-and trans-acting elements allows global transcriptional control of targeted operons.In contrast, Rhizobium meliloti nif genes are regulated by physiological O 2 but not by anabolic N levels. In R. meliloti cultures, since nif gene products are only very weakly expressed and N 2 fixation is not observed, the nif genes probably respond to as-yet-uncharacterized symbiotic signals. While various Rhizobium species all carry a highly conserved NTRB3NTRC cognate pair, its targets do not include nif genes (57)...

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Development of an Efficient Method for Generating and Screening Active Trypsin and Trypsin Variants

Cited by 29 publications

References 35 publications

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris

Substrate specificity of trypsin investigated by using a genetic selection.

Interactive regulation of Azorhizobium nifA transcription via overlapping promoters

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