LACTB E.coli, His

What is LACTB and what is its evolutionary origin?

LACTB is a mammalian active-site serine protein that has evolved from bacterial penicillin-binding proteins (PBPs), which are involved in bacterial cell wall metabolism . It represents a fascinating example of protein evolution, as it is the only PBP homologue found in mammals, having been identified in all mammalian genomes sequenced to date . The protein shares sequence similarity with the beta-lactamase/penicillin-binding protein family of serine proteases, but its function has diverged significantly from its bacterial ancestors . While bacterial PBPs participate in peptidoglycan metabolism, LACTB appears to have acquired novel biochemical properties during eukaryotic evolution, including the ability to form filamentous structures within mitochondria .

Where is LACTB localized in mammalian cells and what is its basic function?

LACTB is primarily localized in the mitochondrial intermembrane space, as demonstrated through multiple complementary experimental techniques including immunofluorescence microscopy, subcellular fractionation, and immuno-electron microscopy . This localization has been confirmed by genetically modified HeLa cells expressing mitochondria-targeted red fluorescent protein, which showed co-localization with LACTB when stained with an anti-LACTB antibody . Further biochemical analyses involving trypsin digestion of digitonin-permeabilized mitochondria revealed that LACTB becomes accessible to proteolytic degradation only after outer membrane solubilization, confirming its intermembrane space localization .

The function of LACTB appears to be quite different from its bacterial ancestors. Remarkably, LACTB polymerizes into stable filaments within the mitochondrial intermembrane space, with lengths extending more than a hundred nanometers . These filamentous structures may play a role in submitochondrial organization and potentially affect mitochondrial metabolon arrangement . Additionally, LACTB has been linked to obesity through gene co-expression analysis, with subsequent validation in transgenic mice where LACTB overexpression resulted in an obese phenotype . While the exact biochemical mechanism for this obesity-promoting effect remains unclear, it suggests that LACTB may be involved in regulating whole-organism energy homeostasis, directly or indirectly affecting metabolic pathways .

What are the most successful expression systems for producing recombinant LACTB?

The expression of recombinant LACTB has been achieved in various systems, with varying degrees of success depending on the specific constructs and conditions employed. Based on the available literature, the most successful expression system appears to be Escherichia coli for mouse LACTB (mLACTB) when expressed as an N-terminal GST fusion protein (GST-mLACTB) . When produced in this manner, full-length GST-mLACTB protein can be recovered using glutathione-agarose affinity chromatography, with its integrity confirmed by MALDI-TOF mass spectrometry and immunoblotting techniques . This approach has successfully yielded properly folded, full-length protein with well-defined secondary structure as demonstrated by Fourier transform infrared spectrometry analysis .

In contrast, when mLACTB was expressed as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag, significant proteolytic degradation of the protein occurred, making it impossible to detect full-length mLACTB . This suggests that the position and nature of the fusion partner significantly influence protein stability during expression. For commercially available LACTB proteins, yeast expression systems have also been utilized, particularly for LACTB from various origins including Rhodobacter capsulatus, Streptomyces lavendulae, and bovine sources . The yeast protein expression system is noted to be an economical and efficient eukaryotic system for both secretion and intracellular expression of proteins .

How do expression conditions affect LACTB yield and integrity?

Expression conditions play a critical role in determining both the yield and structural integrity of recombinant LACTB. Several key factors must be considered when optimizing LACTB expression, particularly when working with E. coli systems. Temperature management during induction is especially important, as lower temperatures (typically 16-25°C) can reduce the rate of protein synthesis, potentially allowing more time for proper folding and decreasing the likelihood of inclusion body formation or proteolytic degradation . The proteolytic degradation observed with certain LACTB constructs, particularly His-tagged versions, suggests that expression conditions must be carefully controlled to minimize protease activity .

The choice of E. coli strain can also significantly impact expression outcomes. Strains lacking certain proteases (such as BL21(DE3) and its derivatives) are generally preferred for recombinant protein expression to minimize degradation . Additionally, induction parameters including IPTG concentration and duration of induction should be optimized for each specific LACTB construct. For N-terminal GST fusion constructs of mLACTB, which have demonstrated successful expression, specific conditions leading to proper folding have been achieved, as confirmed by Fourier transform infrared spectrometry revealing the presence of alpha-helices, beta-sheets and turns consistent with well-defined secondary structure .

What are the challenges in purifying His-tagged LACTB and how can they be overcome?

Purification of His-tagged LACTB presents significant challenges, primarily due to proteolytic degradation. Research has demonstrated that when mLACTB is expressed with either an N- or C-terminal His6-tag, proteolytic degradation occurs, making it impossible to detect full-length mLACTB . This degradation likely occurs during expression and/or purification processes, suggesting that the His-tag may either expose proteolytic sites or interfere with proper protein folding, leading to increased susceptibility to proteases.

To overcome these challenges, several strategies can be implemented. The most successful approach appears to be switching to an N-terminal GST fusion system, which has been shown to yield full-length protein recoverable by glutathione-agarose affinity chromatography . If the His-tag is still preferred for specific downstream applications, researchers might consider:

• Addition of protease inhibitor cocktails during all purification steps

• Reducing purification temperature to minimize protease activity

• Exploring the use of protease-deficient E. coli strains

• Designing constructs with the His-tag positioned differently within the protein sequence

• Incorporating additional fusion partners that might enhance stability

It's worth noting that commercially available LACTB proteins often specify purity levels (typically >90% for yeast-expressed systems and >95-97% for E. coli-expressed systems), suggesting that successful purification is possible with optimized protocols .

What purification methods yield the highest purity of LACTB protein?

The purification method yielding the highest purity of LACTB depends on the expression system and fusion tag employed. For GST-tagged mLACTB, glutathione-agarose affinity chromatography has successfully yielded full-length protein of sufficient purity for downstream analyses including MALDI-TOF mass spectrometry and Fourier transform infrared spectrometry . The purity achieved through this method appears sufficient for structural and functional characterization studies.

For commercially available recombinant LACTB proteins, multiple analytical methods are typically employed to assess purity, including:

• SDS-PAGE analysis, which can verify protein size and relative purity

• Reverse-phase HPLC, which separates proteins based on hydrophobicity

• Immunoblotting with specific antibodies to confirm protein identity

• Mass spectrometry to verify protein integrity and sequence

Purity levels for commercially available LACTB proteins range from >90% for yeast-expressed systems to >95-97% for E. coli-expressed systems . The highest purity (>97%) has been reported for human LACTB (AA 313-547) with His-tag expressed in E. coli, suggesting that with optimized protocols, very high purity can be achieved even with His-tagged constructs .

What techniques are most effective for analyzing LACTB's filament-forming properties?

Analysis of LACTB's unique filament-forming properties requires a multi-technique approach to fully characterize these structures. Electron microscopy (EM) has proven particularly valuable for visualizing LACTB filaments. When extracted intermembrane space proteins are separated by centrifugation in a CsCl-density gradient and examined after negative staining, characteristic filaments composed of globular subunits can be observed at a gradient density of 1.25 to 1.28 g/cm³ . These filaments vary in length, which explains their broad migration pattern during electrophoretic separation .

Whole-mount immuno-electron microscopy with anti-LACTB antibodies has successfully confirmed the identity of these filamentous structures . For in situ visualization of LACTB polymers within mitochondria, specialized preparation techniques for immuno-electron microscopy are required, using nanogold particles coupled to anti-LACTB antibodies . This approach has revealed both individual nanogold particles and larger particle clusters over the mitochondrial intermembrane space .

Complementary biochemical approaches include:

• 2D blue native SDS/PAGE followed by immunoblotting, which has revealed LACTB bands ranging from 600 kDa to several MDa in the native direction of the gel, indicating polymer formation

• Mass spectrometry analysis of gel fractions to confirm the presence of LACTB in high molecular weight complexes

• Density gradient centrifugation to isolate LACTB polymers based on their unique density characteristics

How can researchers effectively analyze the secondary and tertiary structure of LACTB?

Analysis of LACTB's secondary and tertiary structure requires a combination of biophysical techniques. Fourier transform infrared spectrometry has been successfully applied to GST-mLACTB, revealing the presence of alpha-helices, beta-sheets, and turns, consistent with a well-defined secondary structure . This technique provides valuable information about the protein's folding state and can help determine whether recombinant LACTB maintains its native structure during expression and purification.

The tertiary structure of LACTB reveals significant differences compared to other PBP-βLs proteins, particularly in the loops and α-helices surrounding the core structure . Notable structural features include:

• A long L15 loop (residues 467 to 482) formed between β6 and β7 strands

• A helix α4 (residues 227 to 242) that extends parallel to the β-sheet core

• A predicted flexible loop (residues 243 to 290)

These structural elements are not observed in other PBP-βLs proteins and likely contribute to LACTB's unique functional properties . High-resolution structural data has enabled identification of the amino acid motifs (164SISK167, 323YST325, and 485HTG487) that form the catalytic site, including the catalytic S164 residue . The structure also reveals a clearly defined cavity with a predominantly positively charged character, which may represent a binding pocket approximately 20 Å deep .

What experimental approaches can determine LACTB's enzymatic activity?

Determining LACTB's enzymatic activity requires consideration of its evolutionary relationship to beta-lactamases and penicillin-binding proteins while acknowledging its likely functional divergence in mammals. While specific enzymatic assays for LACTB are not extensively detailed in the provided search results, several approaches can be proposed based on the protein's structural and evolutionary characteristics.

Given LACTB's relationship to serine proteases and the identification of its catalytic S164 residue , protease activity assays using fluorogenic or chromogenic peptide substrates would be a logical starting point. These assays could involve monitoring the cleavage of synthetic peptides designed based on potential physiological substrates or general protease substrates. Since LACTB contains a clearly defined cavity with predominantly positive charge , substrate specificity assays using peptides with varying charge distributions could help define LACTB's substrate preferences.

Additionally, given LACTB's mitochondrial localization and potential role in metabolic regulation , metabolomic approaches might be valuable. These could involve:

• Comparing metabolite profiles in mitochondria with normal versus altered LACTB levels

• In vitro assays testing LACTB's activity against potential metabolic substrates

• Enzyme-coupled assays where LACTB activity is linked to a detectable signal through secondary reactions

The connection between LACTB and obesity in transgenic mice suggests that assays measuring effects on lipid metabolism might also be informative for understanding LACTB's functional impacts.

How can researchers investigate the role of LACTB in mitochondrial organization?

Investigating LACTB's role in mitochondrial organization requires methods that can visualize and quantify mitochondrial structural dynamics and correlate these with LACTB's filament-forming properties. Since LACTB forms filaments in the mitochondrial intermembrane space with lengths exceeding a hundred nanometers , techniques that can visualize these structures in situ are particularly valuable.

Super-resolution microscopy approaches such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy could be employed to visualize LACTB filaments and their relationship to mitochondrial membrane organization with resolution beyond the diffraction limit. These could be combined with fluorescent labeling of other mitochondrial components to determine spatial relationships between LACTB filaments and other mitochondrial structures.

Functional approaches might include:

• Genetic manipulation studies where LACTB levels are modulated (overexpression, knockdown, or knockout) followed by assessment of mitochondrial morphology and function

• Expression of mutant LACTB versions with altered filament-forming capabilities to determine the relationship between filament formation and mitochondrial organization

• Biochemical fractionation studies to identify potential LACTB-interacting proteins that might mediate effects on mitochondrial organization

• Live-cell imaging with fluorescently tagged LACTB to monitor dynamic changes in filament formation and mitochondrial structure

The hypothesis that "LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization" could be tested through these approaches, potentially revealing mechanisms by which LACTB influences mitochondrial function.

How can researchers address protein degradation issues when working with recombinant LACTB?

Protein degradation represents a significant challenge when working with recombinant LACTB, particularly with certain construct designs. When mLACTB was expressed as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag, proteolytic degradation occurred, preventing detection of full-length mLACTB . To address these degradation issues, researchers should consider implementing multiple complementary strategies.

First, the choice of fusion partner and its position significantly impacts protein stability. The N-terminal GST fusion approach has proven successful for mLACTB expression, yielding full-length protein recoverable by affinity chromatography . This suggests that researchers should prioritize this configuration when possible. If alternative tags are required for specific applications, comprehensive optimization of expression conditions becomes essential.

Additional strategies to minimize degradation include:

• Reducing expression temperature (16-20°C) to slow protein synthesis and potentially improve folding

• Using protease-deficient E. coli strains such as BL21(DE3) and derivatives

• Including protease inhibitor cocktails throughout all purification steps

• Shortening expression time to minimize exposure to proteases

• Adjusting buffer conditions (pH, salt concentration) to reduce protease activity

• Exploring the addition of stabilizing agents such as glycerol or specific cofactors

For persistent degradation issues, structural analysis of degradation products by mass spectrometry can identify vulnerable regions of the protein, potentially informing the design of more stable constructs through strategic mutation of protease-sensitive sites or domain-based expression approaches.

What strategies can overcome poor solubility or inclusion body formation with LACTB?

While the search results don't specifically mention inclusion body formation for LACTB, this is a common challenge when expressing complex proteins in E. coli and may occur under certain conditions. Several strategies can be employed to enhance LACTB solubility and minimize inclusion body formation during recombinant expression.

The successful expression of soluble N-terminal GST-mLACTB suggests that fusion partners can significantly enhance solubility . GST in particular is known to improve protein solubility through its own high solubility and native folding properties. Other solubility-enhancing fusion partners worth considering include MBP (maltose-binding protein), SUMO, and thioredoxin, which might provide alternatives if GST fusion is not suitable for specific applications.

Additional approaches to improve solubility include:

• Expression at reduced temperatures (16-20°C) to slow protein synthesis and allow more time for proper folding

• Co-expression with molecular chaperones such as GroEL/GroES or DnaK/DnaJ/GrpE systems

• Addition of solubility-enhancing additives to the growth medium such as sorbitol, glycylglycine, or specific metal ions

• Use of specialized E. coli strains designed for improving protein solubility

• Modulation of induction conditions, including lower IPTG concentrations and extended, gentler induction periods

If inclusion bodies still form despite these measures, refolding strategies may be necessary. These could include solubilization in denaturants followed by controlled refolding through dialysis or dilution methods, potentially with additives that promote correct folding such as arginine or specific redox couples to facilitate proper disulfide bond formation if applicable.

How can researchers investigate the relationship between LACTB structure and its filament-forming capabilities?

Investigating the relationship between LACTB's molecular structure and its unique filament-forming capability requires a systematic approach combining structural analysis, mutagenesis, and functional characterization. The 3.1 Å resolution structural data available for LACTB reveals several distinctive structural elements not found in other PBP-βLs proteins, including a long L15 loop (residues 467-482), helix α4 (residues 227-242), and a predicted flexible loop (residues 243-290) . These unique structural features may be critical for LACTB's polymerization properties.

A comprehensive mutagenesis strategy targeting these distinctive structural elements could help identify regions essential for filament formation. Key approaches would include:

• Systematic deletion analysis of specific loops and helices unique to LACTB

• Point mutations of conserved residues at potential protein-protein interaction interfaces

• Domain swapping experiments with related non-filament-forming proteins

• Construction of chimeric proteins combining domains from LACTB with those from related proteins

For each mutant, filament formation could be assessed using the techniques established for wild-type LACTB, including electron microscopy of negatively stained samples, CsCl-density gradient centrifugation, and 2D blue native SDS/PAGE . Correlation of structural alterations with changes in filament formation capacity would help identify the molecular determinants of this unique property.

Additionally, advanced structural biology approaches such as cryo-electron microscopy of LACTB filaments could provide insights into the higher-order organization of these structures, potentially revealing the specific molecular interfaces involved in polymerization.

What approaches can elucidate LACTB's role in metabolic regulation and obesity?

The established link between LACTB overexpression and obesity in transgenic mice points to a significant role for this protein in metabolic regulation, yet the underlying molecular mechanisms remain unclear. Elucidating LACTB's role in metabolism requires a multifaceted approach spanning molecular, cellular, and organismal levels of analysis.

At the molecular level, interactome studies using techniques such as affinity purification coupled with mass spectrometry could identify LACTB-binding partners within mitochondria, potentially revealing connections to metabolic enzymes or regulatory proteins. Metabolomic profiling of mitochondria with normal versus altered LACTB levels could identify specific metabolic pathways affected by LACTB activity. These approaches should focus particularly on lipid metabolism pathways given the obesity phenotype observed with LACTB overexpression.

Cellular studies might include:

• Investigation of mitochondrial respiration, membrane potential, and ATP production in cells with modulated LACTB levels

• Analysis of lipid droplet formation and lipid metabolism in cellular models

• Assessment of mitochondrial morphology and dynamics in relation to LACTB expression

• Examination of interplay between LACTB and key metabolic signaling pathways

At the organismal level, tissue-specific modulation of LACTB expression in animal models could help identify the primary tissues mediating LACTB's metabolic effects. Comprehensive physiological phenotyping including energy expenditure measurements, glucose and insulin tolerance testing, and body composition analysis would provide insights into the specific metabolic parameters affected by LACTB.

The observation that LACTB promotes intramitochondrial membrane organization suggests a potential mechanism whereby LACTB might influence metabolism through effects on the spatial organization of metabolic enzymes within mitochondria. Testing this hypothesis would require correlating LACTB-induced changes in mitochondrial organization with alterations in specific metabolic pathways.

Table 1: Comparison of Expression Systems for Recombinant LACTB Production

Table 2: Analytical Methods for LACTB Structural and Functional Characterization