How to Determine Protein Full-Length Sequence

Determining the protein full-length sequence is a foundational task in protein research, particularly crucial in studies involving novel protein functions, structural analysis, antibody development, and recombinant expression. But how can researchers obtain the complete amino acid sequence of a protein? This article provides a technical overview and practical strategies based on current mainstream approaches and recent advances.

 

What Is a Protein Full-Length Sequence?

A protein full-length sequence refers to the complete amino acid chain translated from the open reading frame (ORF), beginning at the translation initiation site (corresponding to the start codon) and ending at the stop codon. This sequence represents the entire primary structure of a functional protein and forms the basis for both structural and functional studies.

 

How to Obtain the Protein Full-Length Sequence? — Comparison of Common Approaches

1. At the Genetic Level: Starting from mRNA/cDNA

This approach is suitable for known or predicted proteins, where the protein sequence can be inferred from corresponding nucleic acid information.

(1) RT-PCR Combined with Sanger Sequencing

• Procedure: mRNA extraction → reverse transcription into cDNA → design of gene-specific primers → PCR amplification of full-length coding sequence (CDS) → validation by Sanger sequencing

• Applicable Scenario: Target proteins with available predicted sequences (e.g., annotated in NCBI or UniProt)

• Advantages: High accuracy; capable of identifying splice variants

• Limitations: Requires high-quality RNA; may not successfully amplify very long CDS regions

 

(2) RACE (Rapid Amplification of cDNA Ends)

• Function: Complements missing regions at the 5′ or 3′ ends of transcripts, enabling full-length cloning

• Applicable Scenario: Partial sequence is known, but terminal regions are absent

• Recommended Strategy: Use in conjunction with RT-PCR to recover a complete CDS

 

(3) RNA-Seq (Whole Transcriptome Sequencing)

• Advantages: Does not require prior sequence knowledge; enables comprehensive identification of all transcripts

• Analytical Methods: De novo transcriptome assembly or alignment against annotated databases (e.g., RefSeq)

• Applicable Scenario: Non-model organisms or discovery of novel genes

 

MtoZ Biolabs offers high-quality RNA extraction and full-length transcriptome sequencing services to support the construction of complete coding sequences (CDS).

 

2. Protein-Level Identification: Sequence Determination via Mass Spectrometry

In cases where cDNA information is unavailable or only protein samples are present, protein sequence can be inferred through mass spectrometry-based techniques.

(1) Edman Degradation (N-terminal Sequencing)

• Principle: Sequential removal and identification of amino acids from the N-terminus

• Limitation: Applicable to only the first 10 to 30 amino acids; requires a free N-terminal

• Application: Used to verify the N-terminal structure of expressed proteins

 

(2) Enzymatic digestion of proteins combined with LC-MS/MS-based proteomics

• Steps: Proteins are enzymatically digested → LC-MS/MS analysis is performed → Peptide spectra are matched against sequence databases

• Supplementary strategies: de novo sequencing can be applied to directly infer peptide sequences in the absence of a reference database; transcriptomic data integration enables the construction of protein translation frameworks to enhance peptide identification rates.

 

MtoZ Biolabs is equipped with high-resolution mass spectrometry platforms, including the Orbitrap Exploris, supporting high-coverage proteomic profiling and protein sequence assembly.

 

3. Protein Sequence Prediction and Bioinformatics Tools

When DNA or cDNA sequences are available but not yet translated, bioinformatics tools can be employed for sequence derivation and auxiliary analysis:

(1) ORF Finder (NCBI): Identifies open reading frames

(2) Expasy Translate Tool: Translates cDNA sequences into amino acid sequences

(3) SignalP, TMHMM: Predict signal peptides and transmembrane domains to assist in assessing sequence completeness

(4) AlphaFold2 / ESMFold: Utilize structural modeling to indirectly assess protein sequence integrity

 

How to Determine Whether a Sequence is “Full-Length”?

This is a common issue in research. Criteria for identifying a protein full-length sequence may include:

 



 

Accurate determination of the protein full-length sequence is a fundamental and essential step in protein research and expression vector design. MtoZ Biolabs offers a comprehensive, integrated solution by combining high-quality RNA extraction, cDNA cloning, RACE-based sequence extension, and proteomic mass spectrometry to support researchers in protein sequence validation:

1. Full-length coding sequence (CDS) cloning and sequencing

2. RACE amplification and deep transcriptomic sequencing

3. Proteomic mass spectrometry analysis (including support for unknown sequences)

4. Design and validation of expression vectors

 

This integrated approach ensures complete protein sequence information, enabling more efficient and reliable downstream research.

 

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

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Protein Full-Length Sequencing Service