Guide

Recombinant Proteins

Q: When is E. coli expression appropriate for a recombinant proteinase?

When the target is a small domain, when the enzyme does not require disulphide bonds, when activity is not the primary readout, and when very high yield is required. Full-length mammalian proteinases with multiple disulphide bonds and glycosylation typically fail in E. coli.

Q: Why do some recombinant proteins from different manufacturers give different results?

Different expression systems (E. coli vs mammalian give different glycoforms), different construct boundaries, different tag strategies affecting activity, different buffer compositions in the final formulation.

A recombinant protein is any protein produced by expressing a cloned gene — or engineered gene construct — in a heterologous host cell, allowing researchers to obtain defined, reproducible quantities of a protein independent of its native tissue source. The recombinant protein de…

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Chromatography purification of recombinant proteins

What Is a Recombinant Protein? Definition and Core Concepts

The term recombinant protein refers to a protein whose encoding sequence has been introduced into a host organism using recombinant DNA technology. The host — a bacterium, yeast, insect cell line, or mammalian cell line — is referred to as a recombinant cell: a cell carrying foreign genetic material that directs synthesis of the protein of interest. The expressed protein is then isolated, typically purified to defined purity and activity specifications, and supplied as a research reagent.

Three concepts are central to the recombinant protein meaning in a laboratory context:

Defined sequence: The amino acid sequence is known and fixed by the cloned construct, removing the batch-to-batch variability inherent in tissue-derived preparations.
Scalability: Expression in a fermentation or cell-culture system can be scaled to produce milligram or gram quantities that would be impractical to purify from a natural source.
Modifiability: Tags (His, GST, FLAG, Fc), truncations, point mutations, and domain fusions can be engineered into the construct to facilitate purification, detection, or structure-function studies.

What are recombinant proteins, in practical terms? They are the primary positive controls in ELISA and Western blot assays, the substrates and enzymes used in activity and inhibitor-screening assays, the antigens used to raise and validate antibodies, and the structural targets analyzed by X-ray crystallography and cryo-EM. Virtually every modern biochemical and cell-biology workflow touches a recombinant protein at some stage.

How Are Recombinant Proteins Made?

Production of a recombinant protein follows three sequential stages regardless of which host system is used: cloning and construct design, expression, and purification.

Stage 1: Gene Cloning and Construct Design

The coding sequence for the protein of interest is obtained — either by RT-PCR from mRNA, by chemical gene synthesis, or from an existing cDNA library — and subcloned into an expression vector. Key design decisions at this stage include:

Codon optimization: Synonymous codons are adjusted to match the codon-usage bias of the intended host, improving translational efficiency, particularly for mammalian proteins expressed in E. coli.
Affinity tag placement: N-terminal or C-terminal tags (6×His, GST, MBP, Fc) are incorporated to enable affinity chromatography during purification. Tags may be separated from the protein of interest by a protease recognition site so they can be removed downstream.
Signal sequences and secretion: For proteins that are natively secreted or membrane-associated, a signal peptide directing the protein into the secretory pathway is included or swapped for one compatible with the host.
Domain boundaries: Expressing a full-length protein is not always optimal; constructs are frequently designed around catalytic domains, receptor-binding domains, or other functional units defined by published structural or biochemical data.

Stage 2: Expression

The construct is transfected or transformed into the chosen host cell. For transient expression, cells are transfected and harvested within days. For stable expression, clonal cell lines carrying an integrated transgene are generated. Fermentation or bioreactor conditions — temperature, induction timing, dissolved oxygen, feeding strategy — are optimized to maximize yield and solubility of the target protein.

Stage 3: Purification

Cells or conditioned media are harvested and the protein of interest is isolated through one or more chromatographic steps. A typical scheme includes:

Affinity capture (e.g., immobilized metal affinity chromatography for His-tagged proteins, or Protein A for Fc-fusions)
Ion-exchange or hydrophobic-interaction chromatography for further resolution
Size-exclusion chromatography as a final polishing step and to remove aggregates

Final QC typically includes SDS-PAGE, SEC-HPLC for purity and aggregation assessment, endotoxin testing by LAL assay, and a functional assay — most commonly an enzyme activity assay or a binding assay — to confirm biological activity before release.

Common Expression Systems and Their Trade-Offs

Choosing the right host is among the most consequential decisions in recombinant protein production. Each system has a distinct envelope of strengths and limitations relevant to downstream research use.

Expression System	Typical Yield	Post-translational Modifications	Glycosylation	Best For	Key Limitations
E. coli	High (mg–g/L)	Minimal (no glycosylation; limited disulfide formation)	None	Prokaryotic proteins; small domains; proteins not requiring glycosylation; cost-sensitive screens	Inclusion body formation common; endotoxin contamination risk; not suitable for complex disulfide-rich proteins
Yeast (P. pastoris, S. cerevisiae)	Moderate–high	Some eukaryotic modifications; hyper-mannosylation in S. cerevisiae	High-mannose; differs from human pattern	Secreted proteins; proteins needing eukaryotic folding; cost-intermediate projects	Non-human glycan patterns can affect binding or immunogenicity in assays
Insect (Sf9, Hi5 / baculovirus)	Moderate	Good; limited sialylation	Paucimannose; simpler than mammalian	Multi-subunit complexes; kinases; proteins needing correct folding without full human glycosylation	Truncated N-glycans; baculovirus system requires biosafety containment
Mammalian — HEK293	Low–moderate	Full human-type; phosphorylation, acetylation, ubiquitination	Complex human-type N- and O-glycans	Secreted glycoproteins; cytokines; proteins where glycosylation affects activity or receptor binding	Higher cost; longer timelines; lower volumetric yield
Mammalian — CHO	Low–moderate (higher with stable pools)	Full; slight differences from HEK in glycoform distribution	Complex; slight non-human sialic acid content possible	Proteins requiring authentic glycosylation; antibody production; applications sensitive to receptor-binding affinity	Similar cost/timeline constraints to HEK293; stable line generation adds weeks

For proteinases and their inhibitors — the core of Triple Point Biologics' catalog — the choice between E. coli refolded protein, insect-cell-expressed protein, and HEK293-expressed protein is guided primarily by whether the enzyme requires disulfide bonds for activity and whether glycosylation at specific sites affects substrate-binding or inhibitor-interaction data. Many serine and cysteine proteinases are produced as inactive zymogens in the recombinant form and require activation by a defined processing step, a procedure that should be reported in detail within the product's technical data sheet.

What Are the Main Types of Recombinant Proteins?

The question "what are recombinant proteins used for" is best understood by examining the functional categories that dominate catalog and custom production. Recombinant protein types span a wide range of biological functions.

Cytokines and Chemokines

Cytokines — including interleukins, interferons, tumor necrosis factors, and colony-stimulating factors — are low-molecular-weight signaling proteins that regulate immune and inflammatory responses. Because they are active at picomolar to nanomolar concentrations and their activity depends on precise receptor engagement that can be sensitive to glycosylation and correct disulfide pairing, the expression system choice is critical. Mammalian-expressed cytokines are the standard for cell-based functional assays; E. coli-expressed cytokines may be acceptable for antibody-generation antigens or ELISA standards where binding epitopes rather than receptor activity are the priority.

Growth Factors and Receptor Ligands

Growth factors such as EGF, FGF family members, TGF-β isoforms, and neurotrophins are frequently produced in insect or mammalian systems because of their disulfide-rich structures and the importance of correct folding for receptor-binding activity. They are used as proliferation controls in cell culture, as reference standards in binding assays, and as antigens for antibody development.

Enzymes — Including Proteinases

Recombinant enzymes constitute the substrate of core biochemical assays: activity assays measuring kcat and Km, inhibitor screens determining IC50 and Ki, and substrate-profiling experiments using peptide libraries or positional scanning. For proteinases specifically, producing an active, correctly folded enzyme at defined purity — and knowing precisely which residues are included in the expressed construct — is a prerequisite for kinetic data that is reproducible across laboratories. Zymogen constructs that can be activated by a defined processing proteinase are frequently preferred over constitutively active forms because they allow the researcher to control the timing of activation and minimize autolytic degradation during production and storage.

Antibodies and Antibody Fragments

Full-length IgG antibodies, Fab fragments, scFv constructs, and bispecific formats are produced in mammalian systems for research applications ranging from pull-down assays and immunoprecipitation to competition-binding studies. Recombinant antibodies carry defined sequences, making them reproducible lots that do not vary with animal immunization cycles — a significant advantage for long-running research programs.

Structural and Scaffold Proteins

Collagen fragments, extracellular matrix components, viral capsid proteins, and nuclear scaffold proteins are used in structural biology, cell-adhesion assays, and as model substrates for proteinase-cleavage studies. Their large size and repetitive sequences often make heterologous expression challenging, and many are produced as defined domain fragments rather than full-length proteins.

What Are Recombinant Proteins Used For in Research?

The research applications of recombinant proteins fall into several broad and often overlapping categories.

Activity Assays and Inhibitor Screening

A recombinant enzyme at known concentration and defined specific activity is the essential reagent for constructing a reliable enzyme assay. For proteinases, this means a preparation that has been titrated against an active-site standard (such as E-64 for cysteine proteinases or PMSF-related active-site titrants for serine proteinases) so that active enzyme concentration, not total protein concentration, is used in kinetic calculations. Small-molecule inhibitor libraries are then screened against the active enzyme to identify candidates with defined IC50 values, which are subsequently characterized for mechanism of inhibition (competitive, non-competitive, mixed) using Dixon or Cornish-Bowden analysis.

Antibody Validation and Epitope Mapping

Before an antibody is used to detect an endogenous protein in tissue or lysate, its performance should be validated against a known quantity of the recombinant protein. Western blot validation with a recombinant standard of defined molecular weight confirms band identity; ELISA with serial dilutions of recombinant protein establishes the assay's linear detection range. Epitope mapping using overlapping recombinant domain constructs identifies which segment of the protein the antibody recognizes, informing decisions about which isoforms or splice variants the antibody will detect.

Structural Biology

X-ray crystallography, cryo-electron microscopy, and NMR all require milligram quantities of highly pure, homogeneous, correctly folded protein. Recombinant production — particularly in insect and mammalian systems with rigorous final SEC polishing to remove aggregates — is the only practical source for most targets. Selenomethionine labeling for phasing in crystallography is accomplished by growing E. coli expression strains in selenomethionine-supplemented minimal media, a technique unique to bacterial expression.

Drug Screening and Target Engagement Studies

Recombinant proteins serve as the purified target component in biophysical binding assays — surface plasmon resonance, isothermal titration calorimetry, thermal shift assays — used to characterize compound-target interactions. Because these assays measure binding directly rather than through a downstream signal, the purity and homogeneity of the recombinant protein directly influence the reliability of the binding constants determined.

Scientist inspecting a purified protein preparation

How to Choose a Recombinant Protein for Your Experiment

Several parameters should be evaluated when selecting a recombinant protein from a catalog or deciding whether to produce a custom construct.

Species and sequence coverage: Confirm that the expressed construct covers the domain relevant to your assay. A recombinant covering only the catalytic domain will not be an appropriate control for antibodies raised against the prodomain, and vice versa. The start and end residues of the expressed construct should be stated explicitly in the data sheet.
Expression host: Match the host to the application. If your assay depends on glycan-mediated receptor binding, a mammalian-expressed preparation is required. If you are running a fluorogenic peptide cleavage assay where glycosylation is irrelevant, a correctly folded bacterial or insect-cell preparation may be sufficient and more economical.
Tag presence and location: Tags can interfere with active sites, dimerization interfaces, or antibody epitopes. Data sheets should state whether the tag has been removed, and if so, what residues remain after protease cleavage of the tag site.
Purity specification: SDS-PAGE under reducing and non-reducing conditions, SEC-HPLC trace, and endotoxin level (EU/mg) should all be provided. For cell-based assays, endotoxin below 1 EU/µg is a standard threshold; for some cell types, lower thresholds are warranted.
Activity data: For enzymes, a specific activity value — units per milligram, determined with a defined substrate at defined conditions — should be provided and ideally compared to published benchmark values.
Lot-to-lot consistency: Ask whether the supplier holds archived lots or maintains a master cell bank from which consecutive lots are produced, ensuring that an activity change between lots can be traced to a defined production variable.
Availability of a matched antibody: Having a rabbit polyclonal antibody raised against the same protein, validated by the same laboratory, significantly shortens the assay-development cycle. You can confirm detection of endogenous protein using the recombinant as a positive control without worrying about expression-system-specific epitope artifacts.

Triple Point Biologics' Proteinase Catalog: Matched Recombinants and Antibodies

Triple Point Biologics has focused on proteinases and their endogenous inhibitors since 1994. Every recombinant protein in the catalog is produced with a corresponding rabbit polyclonal antibody manufactured in the same laboratory under matched QC protocols — what the catalog describes as a matched-pair offering. The antibodies are raised against defined recombinant antigen fragments (residue ranges stated on each product page), and cross-reactivity predictions are based on sequence identity analysis rather than assumed. Western blot and IHC validation data are provided for each antibody; secondary species reactivity is stated as predicted or validated, not assumed.

This means that when a researcher purchases a recombinant serine proteinase for a substrate-cleavage screen, the rabbit polyclonal raised against that same protein — and validated in Western blot on cell lysates expressing the endogenous enzyme — is available from the same source. The practical benefit is that positive-control bands in Western blot can be run alongside the recombinant standard, and the antibody's epitope location relative to the expressed construct's residue coverage is fully documented.

The catalog covers both active enzymes and inactive zymogen forms where they are available and scientifically meaningful, and includes several proteinase inhibitor proteins (serpins, TIMPs, cystatins) that serve as tool compounds for activity-assay controls.

For targets not currently in the catalog, custom expression and antibody services are available for proteinase family members. Details on production and quality-control methodology are described on the Quality and Validation page.

Frequently Asked Questions

What is the simplest recombinant protein definition?

A recombinant protein is a protein expressed from a cloned or engineered gene in a host cell that does not naturally produce it, allowing controlled, scalable production of a defined protein sequence for research or other applications.

What is a recombinant cell?

A recombinant cell is any cell — bacterial, yeast, insect, or mammalian — that has been stably or transiently introduced with recombinant DNA encoding a gene of interest. In protein production, recombinant cells are used as the expression host that synthesizes the target protein.

How do recombinant proteins differ from native proteins purified from tissue?

Native tissue-purified proteins carry the complete set of post-translational modifications present in the source tissue, which may vary between tissue types, developmental stages, or disease states. Recombinant proteins carry modifications determined by the host expression system, which may partially or fully recapitulate the native modifications depending on the host chosen. The key advantage of the recombinant form is defined sequence, scalability, and lot-to-lot reproducibility; the key trade-off is that some biologically relevant modifications may differ from the native form.

Why does the expression system matter for enzyme activity assays?

For many enzymes, the expression system affects whether the protein folds correctly, forms required disulfide bonds, undergoes necessary glycosylation or phosphorylation, and is produced in a soluble rather than aggregated form. An enzyme expressed in E. coli that requires glycosylation for correct active-site geometry may show lower specific activity than the same sequence expressed in HEK293 cells. Comparing specific activity values against published literature — or against an internal benchmark lot — is the most direct way to confirm that the preparation in hand is appropriately active.

What types of recombinant proteins are most commonly used in proteinase research?

The most common types are: (1) the recombinant proteinase itself, typically as an active enzyme or activatable zymogen; (2) recombinant protein substrates, such as defined domain fragments of extracellular matrix proteins, used to characterize cleavage site specificity; (3) recombinant endogenous inhibitors (TIMPs, serpins, cystatins) used as assay controls or to define inhibitory potency; and (4) recombinant antibody validation standards confirming that the antibody detects the correct molecular weight species under the assay's denaturing or native conditions.

How should I store a recombinant protein?

Storage conditions should follow the manufacturer's data sheet exactly, as optimal conditions are protein-specific. General principles: most purified recombinant proteins are stable at −80 °C in a buffer containing a cryoprotectant (typically 5–10% glycerol), with single-use aliquots prepared before first freeze to avoid repeated freeze-thaw cycles. Enzymes prone to autolysis should be stored with a reversible inhibitor if one is available. Working stocks can frequently be held at 4 °C for short periods (days), but stability data should be confirmed empirically for each lot.

What is the difference between a recombinant protein and a recombinant antibody?

A recombinant antibody is a specific subtype of recombinant protein: an immunoglobulin whose heavy- and light-chain variable region sequences have been cloned and are expressed from defined constructs, as opposed to being produced from a hybridoma or immunized animal. All recombinant antibodies are recombinant proteins, but most recombinant proteins are not antibodies. The term "recombinant protein" in catalog contexts typically refers to non-antibody proteins used as research reagents — enzymes, cytokines, growth factors, and structural proteins.

What information should a recombinant protein data sheet include?

A complete data sheet for a research-use recombinant protein should include: the exact residue range of the expressed construct relative to the canonical UniProt sequence; the expression host and vector tag used; tag removal status; purity by SDS-PAGE (reducing and non-reducing) and SEC-HPLC where available; endotoxin level; specific activity with the substrate and conditions used for the measurement; reconstitution instructions; recommended storage buffer and conditions; and a certificate of analysis cross-referencing the specific lot number. For proteinases, the zymogen or active state of the as-supplied material should be explicitly stated.

Expression system choice — E. coli, insect, HEK293, CHO

Choice of expression system for a recombinant protein is one of the primary design decisions and typically dictates cost, yield, and functional quality. Four systems dominate research-use recombinant protein production, and each is best suited to a subset of protein classes.

E. coli

Best for: Small proteins, protein domains, non-glycosylated targets, high-yield structural biology. Yield: Very high — grams per litre culture achievable. Cost: Lowest. Limitations: No mammalian post-translational modifications, aggregation and inclusion body formation common for complex targets, no disulphide bond formation without engineering (SHuffle strains, PDI co-expression).

Insect cells (Sf9, Sf21, High Five)

Best for: Membrane proteins, large complexes, proteins requiring some post-translational modification. Yield: High — 10-100 mg/L achievable with baculovirus expression. Cost: Moderate. Limitations: Glycosylation is insect-type (paucimannose or high-mannose) not mammalian — inappropriate when specific mammalian glycoforms are required.

HEK293

Best for: Proteinases requiring authentic human folding and glycosylation, therapeutically-relevant targets, quick expression turnaround. Yield: Moderate — 1-100 mg/L with optimized transient transfection. Cost: Higher than E. coli/insect. Limitations: Yields lower than E. coli, host cell protein contamination requires careful purification. This is the primary system for TPB's recombinant proteinase catalog.

CHO

Best for: Stable long-term production, therapeutic-quality proteins, mammalian glycoforms with defined biosimilar characteristics. Yield: High in optimized stable lines. Cost: Higher upfront (stable line generation) but lower per-mg at scale. Limitations: Stable line generation adds 3-6 months to project timeline. Standard for recombinant antibody expression including the CHO-derived recombinant antibodies from TPB's antibody conversion service.

See the protein expression systems comparison guide for the full head-to-head decision framework, and the HEK293 vs CHO guide for the mammalian expression comparison specifically.

Tag choice and purification strategy

Affinity tags simplify purification but affect the final product. Common choices:

His tag (6-His, 10-His): Small, purified on Ni-NTA. Nearly always leaves a small tag remnant after cleavage. Standard for E. coli and initial screening.
GST tag (~26 kDa): Larger, purified on glutathione resin. Can improve solubility but adds substantial mass — cleavage is typically required for functional studies.
FLAG tag (small peptide): Purified on anti-FLAG resin. Small, immunoblot-compatible, moderate cost.
Fc tag (~55 kDa): Purified on Protein A/G. Useful for dimerisation and for surface-plasmon-resonance workflows where the Fc allows oriented immobilisation. Substantial mass addition.
Tag-free / native: Best for functional studies where any tag would perturb activity. Requires alternative purification (ion exchange, hydrophobic interaction, size exclusion in combination).

For proteinases specifically, tag choice affects activity — a tag near the active site or catalytic domain can inhibit or destabilise the enzyme. The convention for TPB's recombinant proteinases is a C-terminal His tag or, where activity requires it, tag-free preparation with alternative purification.

Activity vs structural applications

Applications for recombinant proteins fall into two broad categories: structural/binding applications (where the correct fold matters but catalytic activity is not required) and functional/activity applications (where the enzyme must be active). The distinction dictates which expression system, buffer, and QC standard is appropriate.

Structural / binding applications

Antibody validation (Western blot standard, ELISA standard), surface plasmon resonance binding studies, X-ray crystallography, and NMR structural biology all require the correct fold but not necessarily catalytic activity. Denatured or reduced protein is often adequate for antibody validation; native fold is required for binding and structural work. E. coli-expressed recombinant proteins are frequently sufficient for structural applications.

Functional / activity applications

Enzyme kinetics, activity-based screening, substrate profiling, and inhibitor characterisation all require catalytically active recombinant protein. For most proteinases, this means mammalian expression (HEK293 or CHO) to preserve authentic folding and post-translational modification. Zymogen preparations may require in vitro activation before use; active preparations may require careful storage to prevent autolysis.

Positive-control applications

Recombinant proteinases and inhibitors are the standard positive controls for antibody validation. When a Western blot lane shows a band at the expected molecular weight, the recombinant target run in an adjacent lane confirms that (a) the band is at the correct size, and (b) the antibody detects the recombinant protein — a specificity check. See the recombinant protein positive controls guide for the antibody-validation-specific use case.

Functional assay applications — activity assays and screening

Recombinant proteinases enable activity assays that would be impractical or impossible with tissue-derived enzyme. Common formats:

Fluorogenic substrate assays: A synthetic peptide substrate labelled with a fluorophore-quencher pair produces fluorescence on cleavage. Standard for enzyme kinetics and inhibitor screening. See the fluorogenic substrates guide.
Chromogenic substrate assays: Substrate produces coloured product on cleavage. Standard for coagulation-cascade proteinases (thrombin, factor Xa) and some serine proteases.
Native protein substrate assays: Full-length physiological substrates (collagen for collagenases, β-casein for general proteinases). Higher biological relevance, lower throughput.
Zymography: SDS-PAGE with substrate polymerised in the gel; renaturation and staining reveal clear bands where active proteinase digests the substrate. Standard for MMPs (gelatin zymography).

See the protease activity assays guide for the format-by-format comparison and the protease inhibitor screening guide for the inhibitor-focussed workflow.

Storage and activity preservation

Recombinant proteins — particularly proteinases — are less stable than most other reagent classes and require careful storage. Standard practice:

Aliquot the received lot into single-use volumes on receipt. Repeated freeze-thaw is a primary cause of activity loss.
Store at −80°C in buffer containing 5-10% glycerol as cryoprotectant. Some proteinases are stable at −20°C but −80°C is safer.
For active proteinases, include a reversible inhibitor (EDTA for metalloproteinases, PMSF for serine proteases) if activity is to be recovered rather than measured immediately.
Working stocks at 4°C are viable for hours to days depending on the protein; refer to the CoA for lot-specific stability data.
Log freeze-thaw cycles on the aliquot tube. After 3-5 cycles, expect activity loss.

Choosing between recombinant and native protein

The two alternative sources for research-use protein — recombinant expression and tissue purification — each have specific advantages. In most modern research contexts, recombinant is the correct choice because of defined sequence, batch consistency, and scalability. Tissue-purified is preferable in a few specific cases: when authentic post-translational modification patterns matter and cannot be reproduced in any expression system, when the endogenous target complex (e.g., an enzyme-substrate complex or an enzyme with a bound cofactor) is what is being studied, and when historic literature and comparison data has been generated exclusively with tissue-derived material.

For proteinase research specifically, recombinant is almost always the correct choice. Tissue-purified proteinases are contaminated with other proteinases from the same tissue, batch variability is high, yields are low, and specific activity is difficult to define reproducibly. The primary exception is where the native complex (e.g., an activated enzyme bound to its endogenous inhibitor) is the study object.

Zymogens and pro-enzymes in recombinant catalog

For proteinases, recombinant preparations can be supplied as zymogens (pro-enzymes) or as active enzymes. The choice depends on the application. Zymogens are more stable in storage but must be activated before use; active enzymes are ready to use but subject to autolysis and shorter shelf life. TPB's recombinant proteinase catalog supplies both formats where the enzyme can be reproducibly maintained in either state; refer to individual product pages for the format supplied for a specific target. See the zymogens and pro-enzymes guide for the mechanism-of-activation detail.

Frequently Asked Questions — recombinant protein applications

Should I choose HEK293 or CHO for a recombinant proteinase?

HEK293 for quick expression and moderate yield; CHO for stable long-term production and larger scale. For most research applications, HEK293 is sufficient and preferable because the timeline is shorter. See the HEK293 vs CHO comparison guide.

When is E. coli expression appropriate for a recombinant proteinase?

When the target is a small domain (isolated catalytic domain, single domain fragment), when the enzyme does not require disulphide bonds or when disulphides can be formed post-expression, when activity is not the primary readout, and when very high yield is required. Full-length mammalian proteinases with multiple disulphide bonds and glycosylation typically fail in E. coli.

Do I need to remove the affinity tag for functional experiments?

Depends on the tag and the target. Small tags (His, FLAG) rarely affect activity substantially and are often left in place. Larger tags (GST, Fc) typically require cleavage before functional work. For proteinases where the tag is near the active site or a substrate-binding surface, tag removal is generally required.

How do I test that a recombinant proteinase is active?

Standard activity assay in the appropriate format: fluorogenic substrate for most classes; chromogenic substrate for coagulation-cascade serine proteases; zymography for MMPs; ELISA-based substrate cleavage for lower-throughput profiling. Include a positive control (a well-characterised recombinant proteinase of the same class) and a negative control (buffer only, or the recombinant proteinase pre-inhibited).

What's the difference between a recombinant zymogen and a recombinant active enzyme?

The zymogen is the inactive precursor still carrying the propeptide. The active enzyme has the propeptide cleaved off and the catalytic surface exposed. Zymogens require activation (auto-cleavage, cleavage by an activator protease, or chemical activation such as APMA for MMPs) before catalytic activity is measurable. See the zymogens and pro-enzymes guide.

Can I use recombinant proteinase to raise a custom antibody?

Yes. Customer-supplied recombinant proteins are accepted as immunogens by TPB's custom antibody production service, provided purity and concentration meet minimum requirements. Recombinant protein immunogens produce polyclonals with broader epitope repertoires than peptide immunogens — good for maximum sensitivity, less good for paralogue selectivity.

How pure does a recombinant protein need to be for antibody validation?

For Western blot standard use, >90% purity by SDS-PAGE is sufficient. For ELISA standard use, >95% purity is preferable. For enzyme kinetics work, >95% purity and defined specific activity are required. Refer to individual product CoAs for lot-specific purity data.

Why do some recombinant proteins from different manufacturers give different results?

Common causes: different expression systems (E. coli vs mammalian give different glycoforms), different construct boundaries (a catalytic-domain-only construct vs full-length), different tag strategies affecting activity, different buffer compositions in the final formulation. Compare the CoAs before assuming a preparation is comparable to another.

How do I store a recombinant proteinase to preserve activity?

Aliquot on receipt into single-use volumes. Store at −80°C in a buffer containing 5-10% glycerol. Include a reversible inhibitor if activity needs to be preserved for measurement rather than immediate use. Avoid repeated freeze-thaw. See the storage section above for detailed conditions.

Where does the recombinant catalog fit into TPB's overall product offering?

TPB's recombinant catalog covers proteinases and their inhibitors matched to the antibody catalog — antibody targets are matched by recombinant proteins used for antibody validation, positive controls, and downstream functional work. See the recombinant catalog for the full listing.