Biomedical subjects

M M Ataai

Publications and source records attributed to M M Ataai.

23 records · Page 2Linked to original sources

PubMedDate not supplied

One-step purification of a model periplasmic protein from inclusion bodies by its fusion to an effective metal-binding peptide.

It has been demonstrated that the addition of a metal-binding amino acid sequence to an exposed terminus of a protein can be useful for purification using immobilized metal affinity chromatography (IMAC). Polyhistidine extensions, wherein sequential histidyl residues are placed at the end of a protein, have been utilized for protein purification through IMAC. Natural metal-binding peptides may also serve as starting points for the design of an affinity tail. As a model system, an octapeptide derived from angiotensin I was fused to TEM-beta-lactamase. When the modified protein was expressed in Escherichia coli, a one-step purification of this recombinant protein was accomplished from resolubilized inclusion body material.

Amino Acid Sequence↗

PubMedDate not supplied

Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures: emergence of pyruvate kinase as a potential metabolic engineering site.

Microbial cultures typically produce acids when metabolizing the common carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity. On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective. The effect of citrate on in-vivo flux distribution was quantified using a detailed flux-model. When the molar glucose-citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions. Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.

Bacillus↗

PubMedDate not supplied

Cell growth and by-product formation in a pyruvate kinase mutant of E. coli.

In this paper, we report on the analysis of acid formation in an E. coli pyk mutant. The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations. However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type. This substantial reduction in acids is accompanied by an increase in CO(2) production. Moreover, unlike the B. subtilis pyk mutant, the E. coli pyk mutant did not show a substantial increase in the PEP pool.

Acids↗

PubMedDate not supplied

Design of affinity tags for one-step protein purification from immobilized zinc columns.

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Affinity Labels↗

PubMedDate not supplied

Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.

Calorimetry, Differential Scanning↗