PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Summary

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Abstract

Structural studies with tryptophan synthase (TS) bienzyme complex (α₂β₂ TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated β-subunit allowed the purification of high amounts of pure subunits and α₂β₂ StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme β-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α₂β₂ StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α₂β₂ StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α₂β₂ StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α₂β₂ StTS complexes.

Introduction

The tryptophan synthase (TS) bienzyme complex (α₂β₂) is an allosteric enzyme, catalyzing the last two steps in the biosynthesis of the amino acid L-Tryptophan in bacteria, plants, and fungi^1,2,3. Bacterium Salmonella enterica serovar typhimurium (St) causes a severe gastrointestinal infection in humans and other animals. Since humans and higher animals do not have TS (EC 4.2.1.20), the inhibition of S. typhimurium α₂β₂ TS complex (α₂β₂ St

Protocol

1. Fast protocol to purify the α- and β-subunit and the recombined α₂β₂ StTS complex

1. DNA subcloning into pETSUMO expression vector
   1. Obtain the translationally coupling gene (trpA and trpB) encoding the α- and β-subunits of the tryptophan synthase from bacterium Salmonella enterica serovar typhimurium cloned in the pEBA-10 expression vector²². Use pEBA-10 vector as a DNA template.
      NOTE: Alternatively, the listed primers below can be used to amplify both genes from the Salmonella enterica serovar typhimurium....

Results

Purification of the α- and β-subunits of the tryptophan synthase
The α-subunit (αStTS) and the β-subunit (βStTS) of the Salmonella typhimurium tryptophan synthase were subcloned in the modified pET SUMO vector. Figure 1A shows representative SDS-PAGE results of two strong bands corresponding to the His6-SUMO-αS.......

Discussion

We have successfully engineered mutant form α₂β₂ βQ114A, α₂β₂ βK167T, and α₂β₂ βS377A StTS complexes for structure-function correlation studies. Initially, we have tried to purify the mutants using a previous purification protocol²², which requires α₂β₂ StTS complex crystallization with PEG 8000 and spermine during purification. Although c.......

Materials

Name	Company	Catalog Number	Comments
15 mL 10 kDa filter	MilliporeSigma	UFC901024	centrifugal filter unit
15 mL 100 kDa filter	MilliporeSigma	UFC910024	centrifugal filter unit
2 mL cryogenic vials	Corning	CLS430489	Cryogenic vials
2 mL microcentrifuge tubes	Fisher Scientific	05-408-141	microcentrifuge tubes
24-well Cryschem Plate	Hampton Research	HR3-158	24-well sitting drop plates
2-mercaptoethanol	Fisher Scientific	O3446I-100	Chemical
50 mL centrifuge conical tubes	Thermo Scientific	12-565-270	centrifuge conical tubes
AB15 ACCUMET Basic	Fisher Scientific	13-636-AB15	pH meter
Agarose	Fisher Scientific	BP1356-100	Agarose gel
ammonium sulfate	Fisher Scientific	A702-500	Chemical
Ampicillin	Fisher Scientific	BP1760-5	Antibiotic
Bacterial incubator	Fisher Scientific	S35836	incubator.
BamHI	New England Biolabs	R0136S	Restriction enzyme
bicine	Fisher Scientific	BP2646100	Chemical
Branson 450 Digital Sonifier	Brason	B450	Cell disruptor
Cesium chloride	Fisher Scientific	BP210-100	Chemical
Cesium hydroxide	Acros Organics	AC213601000	Chemical
Chloramphenicol	Fisher Scientific	BP904-100	Antibiotic
dimethyl sulfoxide	Fisher Scientific	D1391	Chemical
dithiothreitol	Fisher Scientific	BP172-5	Chemical
DNA Polymerase	Thermo Scientific	F530S	HF polymerase
dNTP Set	Invitrogen	10-297-018	dNTPs set
EcoRI	New England Biolabs	R0101S	Restriction enzyme
Ethylenediaminetetraacetic acid	Fisher Scientific	S311-100	Chemical
Excella E25R Orbital Shaker	Eppendorf New Brunswick	M1353-0004	Orbital incubator
GE AKTA Prime Plus	GE Healthcare	8149-30-0004	FPLC
Gel Extraction Kit	Invitrogen	K210012	DNA purification kit
Glycerol	Fisher Scientific	G33-500	Chemical
HindIII	New England Biolabs	R0104S	Restriction enzyme
His-Trap columns	GE Healthcare	GE17-5255-01	5 mL Histrap column
imidazole	Fisher Scientific	O3196-500	Chemical
IPTG	Thermo Fisher Scientific	R0392	Inducer
Kanamycin	Fisher Scientific	BP906-5	Antibiotic
Kelvinator Series-100	Kelvinator	discontinued	Ultra low freezer
LB broth	Fisher Scientific	BP1426-500	Liquid broth
Luria Bertani agar	Fisher Scientific	BP1425-2	Solid broth
NaCl	Fisher Scientific	S271-500	Chemical
NcoI	New England Biolabs	R0193S	Restriction enzyme
Ni-NTA affinity beads	Thermo Fisher Scientific	R90115	Ni-NTA agarose beads
PEG 8000	Fisher Scientific	BP233-100	Chemical
phenylmethylsulfonyl fluoride	Fisher Scientific	44-865-0	Chemical
pyridoxal phosphate	Acros Organics	AC228170010	Chemical
S-200 HR	Cytiva	45-000-196	Size exclusion column
SacI	New England Biolabs	R0156S	Restriction enzyme
Sodium hydroxide	Fisher Scientific	S318-100	Chemical
Sorvall RC-5B centrifuge	Sorvall	8327-30-1004	Floor cetrifuge
Spermine	Acros Organics	AC132750010	Chemical
Superdex 200 prep grade	Cytiva	45-002-491	Size exclusion column
T4 DNA ligase	New England Biolabs	M0202S	DNA liagse
Tris	Fisher Scientific	BP152-500	Chemical
Ubl-specific protease 1	Thermo Scientific	12588018	SUMO Protease