Part:BBa_K3742002

pET16b-L593 and pET28a-hCOL3 > gene sequence

Description

In this part, we designed a plasmid containing a fragment of human collagen type III COL3A1cDNA(hCOL3). We planned to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to modify collagen encoded by hCOL3 to produce hydroxylated collagen, which can provide a better cell culture environment for cultured meat.[1]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1129
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 373
  Illegal AgeI site found at 814
  
• 1000
  COMPATIBLE WITH RFC[1000]

I. Purpose of the experiment

Collagen is a family of proteins widely distributed in animal connective tissue, and it is also the most abundant and widely distributed functional protein in mammals. Because of its good physical and biological properties, it is widely used in medicine, tissue engineering, food and other fields. Type Ⅲ collagen is a homogeneous trimer formed by three α1 chains, which can provide sufficient nutrients for cells and make the skin full and bright. It is often used as a scaffold for tissue engineering[2].
Our experimental group planned to provide a better cell culture environment for cultured meat by expressing hydroxylated collagen. We selected the expression of collagen hCOL3 and L-593 proline hydroxylase (Hydroxylated proline can significantly improve the anti-degradation ability of peptides.) in E.coli (BL21) respectively(considering the stability and activity of expression in E. coli), then carried out enzyme modification reaction in suitable and vitro environment, finally, purified the hydroxylated collagen and determined its content[3].

II. pET16b-L593 and pET28a-hCOL3 > plasmids

We found that some aquatic giant viruses, belonging to Phycodnaviridae and Mimiviridae, harbor genes encoding prolyl hydroxylase enzyme[4]. These viral hydroxylases are soluble and active when expressed in E. coli, thus opening new possibilities for the production of hydroxylated collagen in bacterial expression systems[5].We planed to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to produce hydroxylated collagen. For figure, it is the pET16b-L593 vector that we synthesized uses pET-16b as the expression vector and lacks His-Tag.

Primers and restriction Enzyme cutting sites:
5’-CCATGGATGAAAACTGTAACAATAATCACG-3’
5’-CGAACGTAAGTTCAGCTAAGGATCC-3’

Tips:
(1) The red part is T7 promoter:
(2) The green part is Prolyl 4-hydroxylase [Acanthamoeba polyphaga mimivirus] gene (NCBI Reference Sequence: YP_003987108.1）
(3) The blue part is T7 terminator

Protein sequence:
MKTVTIITII VVIIVVILII MVLSKSCVSH FRNVGSLNSR DVNLKDDFSY ANIDDPYNKP
FVLNNLINPT KCQEIMQFAN GKLFDSQVLS GTDKNIRNSQ QMWISKNNPM VKPIFENICR
QFNVPFDNAE DLQVVRYLPN QYYNEHHDSC CDSSKQCSEF IERGGQRILT VLIYLNNEFS
DGHTYFPNLN QKFKPKTGDA LVFYPLANNS NKCHPYSLHA GMPVTSGEKW IANLWFRERK
FS

We chose to use a fragment of human collagen type III COL3A1cDNA, encompassing 1206 bp and lacking propeptide-encoding regions, was custom synthesized using codons optimized for bacterial expression and including NcoI and BamHI sites at 5’- and 3’-ends. The pET28a expression vector was first digested with NcoI-BamHI, which eliminates the His-tag at the 5N-terminal site. The resulting hCOL3 segment was inserted as a NcoI-BamHI fragment into pET28a, yielding pET28a-hCOL3 with His-tag.
Primers and restriction Enzyme cutting sites:
NcoI 5’-CCATGGATGTACGACAGTTATG-3’
BamHI 5’-CCACCATCACCACTAAGGATCC-3’

Tips:
(1) The red part is T7 promoter:
(2) The blue part is synthetic construct for human collagen gene, type III, alpha 1 (COL3A1 gene) fragment(hCOL3)(GenBank: HG779440.1);
(3) The yellow part is 6xHis tag gene sequence;
(4) The purple part is T7 terminator.

Protein sequence:

Tips:
(1) The green part is synthetic construct for human collagen, type III.
(2) The red part is 6xHis tag.

III. Experimental results and analysis

1.SDS protein electrophoresis of expression of L593

We put pET16b-L593 into E. coli (BL21) and induced expression by adding IPTG at 18 °C and 30 °C.Through SDS protein electrophoresis, and comparing with marker, we can confirm the expression of L593.

2.SDS protein electrophoresis of purified hydroxylated collagen and content determination of hydroxylated collagen

The expressed hCOL3 protein and L593 enzyme were carried out enzyme modification reaction a suitable and vitro environment to obtain hydroxylated collagen. Because it has His-tag, it can be purified. The following picture shows SDS protein electrophoresis of purified hydroxylated collagen and content determination of hydroxylated collagen.
(Temperature-The times of elution product)

Through SDS protein electrophoresis, and comparing with marker, we can confirm the expression and purification of hydroxylated collagen.

The picture shows the content of hydroxylated collagen determined by coomassie blue staining method.

References

[1]史静静,高源,贺婧,等. 毕赤酵母中人Ⅲ型胶原蛋白α链与病毒羟脯氨酸酶的共表达[J]. 西北大学学报（自然科学版）,2017,47(2):231-236. DOI:10.16152/j.cnki.xdxbzr.2017-02-014.
[2]Gineyts E，Cloos Pa,Borel o，ef a1．Raeemizatinn and isomerization of type I collagen C--telopeptides in human bone andsot}t tissues：assessment of tissue tllrnovel-．Biochemical．2000，345：481-485．
[3]Rutschmann C;Baumann S;Cabalzar J;Luther KB;Hennet T; “Recombinant Expression of Hydroxylated Human Collagen in Escherichia Coli.” Applied Microbiology and Biotechnology, U.S. National Library of Medicine, https://pubmed.ncbi.nlm.nih.gov/24362857/.
[4]Eriksson M, Myllyharju J, Tu H, Hellman M, Kivirikko KI (1999) Evidence for 4- hydroxyproline in viral proteins. Characterization of a viral prolyl 4-hydroxylase and its peptide substrates. J Biol Chem 274(32):22131-4
[5]Luther KB, Hulsmeier AJ, Schegg B, Deuber SA, Raoult D, Hennet T (2011) Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme. J Biol Chem 286(51):43701-9