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

Abstract

Structural studies with tryptophan synthase (TS) bienzyme complex (α 2 β 2 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 αand isolated β-subunit allowed the purification of high amounts of pure subunits and α 2 β 2 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, sitedirect mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α 2 β 2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α 2 β 2 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 α 2 β 2 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 α 2 β 2 StTS complexes.

R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils

“…We analysed LRRK2 dependent phosphorylation of pRab10 Thr73 in peripheral blood neutrophils from 42 LRRK2 mutation carriers in comparison to 32 controls as well as 27 iPD patients. Peripheral blood neutrophils were isolated from fresh blood by immunomagnetic negative selection as previously described [ 12 ]. Prior to cell lysis neutrophils were split in half and treated with and without the specific LRRK2 kinase inhibitor MLi-2 to demonstrate that pRab10 Thr73 phosphorylation is mediated by the LRRK2 kinase.…”

“…We, therefore, conclude that the effect of the LRRK2 G2019S mutation in heterozygosity as with the participants in our study coupled with the biological variation in between human samples is too small to accurately yield a significant difference by quantitative multiplexed immunoblotting or even more sensitive mass-spectrometry methodologies. On the other hand, measuring pRab10 Thr73 phosphorylation levels using our relatively straightforward immunoblotting assay [ 12 ] may be attractive for researchers without access to a mass-spectrometry facility as the data obtained was similar in quality and resolution to the mass-spectrometry data.…”

“…Peripheral blood neutrophils were isolated directly from fresh blood using EasySep Direct Human Neutrophil Isolation Kit (Stemcell Technologies, Cat# 19,666) based on an immunonegative magnetic selection process as described before [ 11 , 12 ]. Pure and alive neutrophils were then pelleted by centrifugation at 335 g for 5 min and resuspended in 20 ml RPMI 1640 media.…”

“…Serine 935 phosphorylation of LRRK2 is one of the principal pharmacodynamic markers for in vivo LRRK2 kinase inhibition [ 15 ], but not for LRRK2 kinase activity. More recently, the discovery of a subgroup of RabGTPases, including Rab10, as endogenous LRRK2 kinase substrates that are phosphorylated at a conserved Threonine or Serine in conserved switch II domains [ 39 ], the availability of relevant tools [ 24 ] and technologies [ 21 ] as well as the exploration of novel biomatrices [ 12 ] has opened up new opportunities to assess how LRRK2 mutations impinge on is kinase activity.…”

“…Phosphorylation of Rab10 at Threonine 73 (pRab10 Thr73 ) is quantified by either immunoblot analysis deploying highly sensitive monoclonal phosphospecific antibodies raised against the pRab10 Thr73 epitope [ 11 ] or by a targeted mass-spectrometry approach [ 21 ]. As a biomatrix, peripheral blood neutrophils lend themselves for the study of the LRRK2 pathway as they constitute a homogenous and abundant pool of cells that contain relatively high protein copy numbers of both LRRK2 and Rab10 [ 11 , 12 ]. We have recently utilized this assay to demonstrate that LRRK2 dependent pRab10 Thr73 phosphorylation is significantly increased in PD patients carrying a heterozygous VPS35 D620N mutation that augments LRRK2 kinase activity by a yet unknown mechanism.…”