Proline hydroxylase alpha subunit mutants and expression systems thereof

Abstract

This invention relates to the construction of a human placental-derived proline hydroxylase α subunit mutant and its expression vector. Through analysis and screening of the natural hydroxylase subunit, a mutant protein and its construct that are efficiently expressed in Ecoli BL21(DE3) with significantly improved hydroxylation efficiency were obtained.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering, protein engineering, and biomedicine, specifically relating to a method for improving the structural stability, bioactivity, and functionality of recombinant collagen prepared in prokaryotic expression systems such as *E. coli* through in vitro enzyme modification technology via post-translational modification. This invention particularly focuses on the engineering of proline hydroxylase (P4H) and its application in the in vitro modification of recombinant collagen, aiming to address the lack of natural post-translational modification capabilities in prokaryotic expression systems. Background Technology

[0002] High-quality recombinant collagen has become an important alternative to animal-derived collagen due to its advantages such as no viral risks, no immunogenicity, and high designability, and it has broad application prospects in medical devices, cosmetics, and tissue engineering. Currently, *E. coli* is widely used as an efficient and economical expression system for the production of recombinant proteins.

[0003] However, prokaryotic expression systems such as *E. coli* naturally lack complex post-translational modification mechanisms, especially the hydroxylation modifications specific to collagen (mainly proline and lysine hydroxylation). These hydroxylation modifications are crucial for the formation of a stable triple helix structure in collagen and are essential for maintaining its biological function. Recombinant collagen lacking sufficient hydroxylation modifications typically exhibits lower thermal stability, enzyme resistance, and biological activity compared to natural collagen.

[0004] In existing technologies, eukaryotic expression systems (such as yeast cells, insect cells, or mammalian cells) are commonly used to achieve post-translational modification of recombinant collagen. However, these systems suffer from drawbacks such as high production costs, low expression levels, complex purification processes, and susceptibility to viral contamination. Therefore, developing a method for efficiently expressing recombinant collagen in Escherichia coli and then effectively and specifically modifying it in vitro has become a pressing technical problem to be solved in this field. Summary of the Invention

[0005] This invention aims to provide a method that addresses the problem of ineffective post-translational modification of recombinant collagen by an E. coli expression system through an in vitro enzyme modification strategy. This significantly improves the structural stability, thermal stability, and biological functionality of recombinant collagen, making it more consistent with the characteristics of natural collagen, while maintaining the cost-effectiveness and production efficiency advantages of prokaryotic expression systems.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0007] A proline hydroxylase α subunit mutant, characterized in that the mutant introduces mutations at positions H429, D431 and / or R396 on the basis of natural P4HA1.

[0008] Furthermore, the mutations are H429Y, D431E, and / or R396K.

[0009] Furthermore, the mutation further includes Y426F and / or S410L.

[0010] Another aspect of the present invention relates to a proline hydroxylase α subunit mutant, characterized in that the mutant is based on the natural P4HA1 and introduces the Y426F and / or S410L mutations.

[0011] Preferably, the present invention relates to a proline hydroxylase α subunit mutant, characterized in that the mutant is based on the natural P4HA1 and undergoes the following mutations: H429Y, D431E, R396K, Y426F and S410L.

[0012] On the other hand, the present invention also relates to a fusion protein expression system, characterized in that a SUMO tag and a GB1 tag are simultaneously introduced into the pET-28a expression vector.

[0013] Preferably, the expression vector consists of, from 5' to 3', the following sequence: SUMO tag - Linker - proline hydroxylase α subunit mutant - Linker - GB1 tag - His6 tag.

[0014] Preferably, the Linker is (Gly4Ser)3.

[0015] This invention also relates to the application of the above-mentioned proline hydroxylase α subunit mutant and expression system in the post-translational modification of recombinant collagen.

[0016] The technical advantages of this invention are:

[0017] Based on AlphaFold structure prediction, five key mutations (R396K / S410L / H429Y / D431E / Y426F) were introduced simultaneously in the P4HA1 subunit for the first time, overcoming the limitations of natural enzymes and achieving a hydroxylation efficiency of >92% (compared to 58% for wild type).

[0018] The construct of this invention uses the SUMO-GB1 dual-tag system (N-terminal SUMO + C-terminal GB1), which significantly improves the expression level and solubility of P4HA1 mutant in human poorly soluble protein expression platform (E. coli BL21(DE3)). Attached Figure Description

[0019] Figure 1 Comparison of Michaelis-Menten curves;

[0020] Figure 2Comparison of HYP modification efficiency Detailed Implementation

[0021] The following embodiments are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of the present invention, but the scope of protection of the present invention is not limited thereto. This section provides a more detailed description of the present invention in conjunction with specific implementation examples, and its technical features and advantages will be clearly demonstrated in the description. It should be noted that the embodiments described are merely illustrative examples and are not intended to limit the scope of the claims of the present invention. Any detailed adjustments, equivalent substitutions, or adaptive improvements based on the core principles of the present invention fall within the substantive protection scope of the present invention.

[0022] Example 1: Enzyme Modification Mutation

[0023] This study engineered a human placental-derived proline hydroxylase (P4H) α-subunit (P4HA1, NCBI: NM_000917). This natural enzyme forms a heterotetramer with the β-subunit (P4HB, P07237), but its application is limited by the following: 1) High cofactor dependence: [Further details needed for complete translation]. + 1) Sensitive to 2-oxoglutaric acid (2-OG) concentration; 2) Insufficient stability: easily inactivated and poor thermal stability; 3) Low expression efficiency: easily forms inclusion bodies in prokaryotic systems.

[0024] Using AlphaFold structure prediction and PyMOL analysis (refer to PDB: 3GZE), key functional sites were identified, including key metal and cofactor binding sites (conserved residues) in the P4HA1 subunit and residues near the substrate binding region that may affect substrate affinity and triple helix adaptability, as shown in Tables 1 and 2:

[0025] Table 1 Key metal and cofactor binding sites (conserved residues) in the P4HA1 subunit

[0026]

[0027] Table 2. Residues near the substrate binding region that may affect substrate affinity and triple helix adaptability.

[0028]

[0029] The amino acid sequence of natural P4HA1 (NCBI: NM_000917) is as follows:

[0030] MIWYILIIGILLPQSLAHPGFFTSIGQMTDLIHTEKDLVTSLKDYIKAEEDKLEQIKKWAEKLDRLTSTATKDPEGFVGHPVNAFKLMKRLNTEWSELENLVLKDMSDGFISNLTIQRQYFPNDEDQVGAAKA LLRLQDTYNLDTDTISKGNLPGVKHKSFLTAEDCFELGKVAYTEADYYHTELWMEQALRQLDEGEISTIDKVSVLDYLSYAVYQQGDLDKALLLTKKLLELDPEHQRANGNLKYFEYIMAKEKDVNKSASDDQS DQKTTPKKKGVAVDYLPERQKYEMLCRGEGIKMTPRRQKKLFCRYHDGNRNPKFILAPAKQEDEWDKPRIIRFHDIISDAEIEIVKDLAKPRLSRATVHDPETGKLTTAQYRVSKSAWLSGYENPVVSRINMR IQDLTGLDVSTAEELQVANYGVGGQYEPHFDFARKDEPDAFKELGTGNRIATWLFYMSDVSAGGATVFPEVGASVWPKKGTAVFWYNLFASGEGDYSTRHAACPVLVGNKWVSNKWLHERGQEFRRPCTLSELE

[0031] Three primer pairs were designed using a strategy based on overlap PCR or site-directed mutagenesis to introduce five mutations: R396K (CGA→AAA), S410L (TCC→CTG), Y426F (TAT→TTT), H429Y (CAT→TAT), and D431E (GAC→GAA). The primer pair sequences are shown in Table 3 below.

[0032] Table 3. Mutant Primer Sequences

[0033]

[0034] The study employed a stepwise cumulative mutation strategy, first constructing R396K, then introducing S410L, and finally introducing Y426F, H429Y, and D431E, resulting in sequences containing five mutation sites. Sanger validation or cloning validation was performed: the PCR product was cloned into pET-28a(+) using restriction endonucleases (NdeI / XhoI), and 10 single clones were selected for sequencing validation.

[0035] Example 2 Construction of mutant enzyme expression system

[0036] To efficiently express the P4HA1 mutant in E. coli BL21(DE3), this invention uses the pET-28a(+) expression vector to construct a fusion protein system consisting of an N-terminal SUMO tag + flexible linker + P4HA1 mutant + flexible linker + C-terminal GB1 tag + His6 tag, with the specific structure as follows: 5' → [SUMO] - [(Gly4Ser)3Linker] - [P4HA1 mutant] - [(Gly4Ser)3Linker] - [GB1] - [6×His] → 3'.

[0037] The functional descriptions of each component of the constructed P4HA1 fusion expression vector are shown in Table 4:

[0038] Table 4 Functional descriptions of each component of the P4HA1 fusion expression vector

[0039]

[0040] Specifically, the functions of each tag are explained below:

[0041] SUMO tag (N-terminus): Enhances expression and solubility, possesses molecular chaperone activity. SUMO (Small Ubiquitin-like Modifier) ​​is a widely used tag for expressing poorly soluble proteins in prokaryotic systems, exhibiting excellent hydrophilicity and structural stability. When located at the N-terminus of the target protein, it can prevent the rapid aggregation of nascent polypeptide chains into inclusion bodies during translation through a "dissolution-guided" effect. SUMO possesses molecular chaperone-like function, assisting the target protein in folding into the correct spatial conformation, reducing misfolding and degradation. SUMO can be efficiently recognized and cleaved by specific SUMO proteases (such as Ulp1), with precise cleavage sites, enabling the recovery of the native target protein sequence without any tag residue.

[0042] GB1 tag (C-terminal): Enhances stability, improves expression levels, and facilitates purification. GB1 is the B1 domain of human immunoglobulin G, with a compact structure and stable expression. As a C-terminal tag, it can significantly improve the translation efficiency and overall expression level of fusion proteins. GB1 also has good hydrophilicity and synergizes with SUMO, helping to reduce the formation of inclusion bodies by the expression product. GB1 can specifically bind to IgG, providing an additional means for the selective removal of P4HA1 mutants in subsequent hydroxylase systems (such as capture via IgG magnetic beads / affinity columns), thereby improving the purity of collagen products.

[0043] SUMO and GB1 are located at the N-terminus and C-terminus, respectively, providing support in stages such as translation initiation, fold protection, steric stabilization, and expression release. SUMO alleviates early aggregation of nascent peptide chains, while GB1 enhances expression efficiency and stability in later stages of translation. This combination of "leader protection + back-end support" helps to significantly reduce inclusion body formation, increase the proportion of soluble proteins, enhance protein stability in vivo, reduce degradation, and facilitate the acquisition of high-purity, functional expression products.

[0044] Example 3 Expression and purification of mutant enzymes

[0045] E. coli BL21(DE3) was transformed, and expression was induced by IPTG. Cells were lysed (by sonication / enzymatic digestion), and the supernatant or inclusion bodies were collected and subjected to affinity chromatography (Ni-NTA purification of His6-tagged protein). The SUMO tag was removed using SUMO protease, while GB1 was retained. Protein purity and size were verified by SDS-PAGE.

[0046] Example 4 Quality assessment of mutant enzymes

[0047] Refolded human recombinant collagen (1-5 mg / mL) was mixed with engineered P4HA1 mutant (enzyme activity ≥50 U / mg) at a ratio of 10:1 (w / w). The reaction system contained:

[0048] 1) Cofactor: 0.1 mM Fe² + (French sulfate, freshly prepared), 2 mM ascorbic acid (to maintain Fe²) + Reduced state), 1 mM α-ketoglutarate (2-OG);

[0049] 2) Buffer solution: 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.01% Tween-20 (to reduce non-specific adsorption);

[0050] 3) Reaction volume: 1-10 mL (adjust according to the scale of the experiment).

[0051] 4) Reaction conditions and monitoring: The reaction was carried out at 35°C and pH 7.5±0.2 for 4-5 hours. The reaction endpoint was determined by real-time monitoring of 2-OG consumption using HPLC. At the end of the reaction, 10 mM EDTA was added and the mixture was placed in an ice bath.

[0052] Amino acid analysis and high-performance liquid chromatography (HPLC) of the modified recombinant collagen showed that the substrate affinity of the mutant enzyme was significantly enhanced, with its KD value for collagen tripeptide decreasing from 12 μM in the wild type to 2.5 μM. Figure 1This indicates a 4.8-fold increase in substrate binding efficiency, demonstrating that the mutant enzyme exhibits a higher reaction rate under low substrate concentration conditions (<10 μM); regarding hydroxylation efficiency, the proline hydroxylation rate (HYP) significantly increased from 58% in the wild type to over 92%. Figure 2 ).

Claims

1. A human placental-derived proline hydroxylase mutant, characterized in that, The α subunit and β subunit of the mutant form a heterotetramer, wherein the α subunit is mutated from the natural P4HA1 as follows: H429Y, D431E, R396K, Y426F and S410L.

2. A mutant expressing the proline hydroxylase of claim 1, characterized in that, The pET-28a expression vector is simultaneously introduced with a SUMO tag and a GB1 tag. The expression vector is composed of the following tags from the 5' to 3' ends: SUMO tag - Linker - the proline hydroxylase α subunit mutant of claim 1 - Linker - GB1 tag - His6 tag. The Linker is (Gly4Ser)3.

3. The application of the proline hydroxylase mutant and expression system according to claims 1-2 in the post-translational modification of recombinant collagen.

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