Recombinant Expression of Human Antimicrobial Peptides,LL-37/Dermcidin, in Microbial Systems = 미생물발현계를 이용한 인간유래 항미생물펩타이드 LL-37과 Dermcidin의 생산

다국어 초록 (Multilingual Abstract)

Rapid spread of antibiotic resistance in microorganisms has been a driving force in search for new classes of antibiotics possessing novel modes of action as well as different cellular targets compared to existing antibiotics in order to decrease the likelihood of development of resistance. The antimicrobial peptides (AMPs) are good candidates for new class of antibiotics, produced naturally by various organisms, including bacteria, plants, insects, fish, and mammals. Although they have different amino acid composition, most of them share similar features, such as a net positive charge and the ability to form amphipatic α-helix or β-sheet structure in which clusters of hydrophobic and hydrophilic amino acids are spatially separated.
The human cationic antimicrobial protein of 18 kDa (hCAP-18) belongs to the class of cathelicidins. It is released from activated neutrophil granulocytes. After release, the 37-amino acid α-helical C-terminal end is cleaved off, forming the functional antimicrobial peptide LL-37. Since LL-37 is the only human antimicrobial peptide in the cathelicidin family and shows a broad spectrum of antimicrobial activity, there is a significant interest in developing this peptide for pharmaceutical applications. Dermcidin (DCD) is a recently discovered AMP with no homology to other known AMPs. Full length product of the dermcidin gene consists of 110 amino acid residues with an N-terminal 19 amino acid signal peptide characteristic for secreted proteins. It is proteolytically processed to form C-terminal peptides of 48 amino acid residues (DCD-1L), 47 amino acid residues (DCD-1), and shorter fragments. These two peptides have a broad spectrum of antimicrobial activity over a broad pH range and in high salt concentrations. These characteristics make it viable alternatives to conventional-synthetic antibiotics.
Antimicrobial peptides with a short length of amino acid sequence can be prepared by a solid phase chemical synthesis, but it could be very expensive. On the other hand, recombianant method using microorganism as host could be an alternative choice. In this study, both LL-37 and DCD-1L gene were cloned from human fetal cDNA library and were expressed as soluble, active form of LL-37 and DCD-1L in Escherichia coli or Pichia pastoris. Furthermore, expressed DCD-1L peptide in E.coli, was purified by one step chitin column.

국문 초록 (Abstract)

인류가 항생제를 사용하고, 사용량이 증가함에 따라서 항생제 저항성 균주가 급증하였고, 기존 항생제와 구분되는 새로운 작용기작을 가진 항생제를 필요로 하게 되었다. 이런 관점에서, 항미생물펩타이드는 새로운 항생제로 사용되기에 충분하다. 항미생물펩타이드는 세균, 식물, 어류 및 포유동물등의 다양한 생물에서 생산된다. 비록 각각의 항미생물펩타이드가 다른 아미노산 서열로 구성되어 있으나, 대부분은 net charge가 양의 값을 가지고 양극성의 α-helix 혹은 β-sheet구조를 이루며, 친수성과 소수성 클러스터가 분리되어 있다는 공통점을 가진다.
인간유래의 18kDa 크기의 양이온성 항미생물 단백질(hCAP-18)은 cathelicidin에 속하며, 활성화된 호중구(백혈구의)에서 생성된다. 이후 C-말단의 37개의 아미노산이 분리되어 활성을 가진 항미생물펩타이드인 LL-37이 된다. LL-37이 인간에게 유일한 cathelicidn이고 폭넓은 항미생물활성능을 보이는 이유 때문에, 이 펩타이드의 의약적 응용에 대한 많은 관심이 모아지고 있다. Dermcidin(DCD)은 최근에 발견된 항미생물펩타이드로 이전에 알려진 다른 항미생물펩타이드와 상동성이 없다. Dermcidin 유전자에서 만들어진 단백질은 분비단백질의 특징인 N-말단의 19개의 분비신호 아미노산을 포함한 110개의 아미노산으로 구성되어 있으며, 이후 C-말단의 48개의 아미노산(DCD-1L), 47개의 아미노산(DCD-1), 그리고 더 짧은 아미노산으로 변형된다. 이러한 LL-37과 Dermcidin, 두개의 펩타이드는 넓은 항미생물능을 가지며 넓은 pH범위와 높은 염 농도에서 활성을 유지 한다. 이러한 특성은 상용화된 합성 항생제를 대체하는데 사용될 수 있다.
짧은 길이의 아미노산 서열로 구성된 항미생물펩타이드는 고체상 합성법으로 합성될 수 있으나, 비용이 많이 드는 단점을 가진다. 때문에 미생물을 이용한 재조합단백질 생산법이 대안으로 사용될 수 있다. 본 연구에서는 LL-37과 DCD-1L펩타이드를 인간 태아 cDNA library에서부터 클로닝하고, 활성형의 LL-37과 DCD-1L을 대장균 혹은 Pichia pastoris발현계를 사용하여 발현하였다. 또한 DCD-1L펩타이드는 chitin column을 사용하여 정제하였다.

목차 (Table of Contents)

• CHAPTER Ⅰ Molecular cloning, expression, and purification of Human AMP, LL-37 and Dermcidin, in E.coli = 14
• Abstract = 15
• Introduction = 16
• Materials and Methods = 23
• Strains, plasmids, culture media and Recombinant DNA techniques = 23
• PCR amplification and cloning of the human LL-37 and Dermcidin coding sequences = 24
• Nucleotide sequence analysis = 25
• PCR amplification using specific primers and subcloning = 25
• Purification of recombinant peptides = 28
• NanoLC-ESI-MS/MS analysis for protein identification = 29
• Antimicrobial analysis of recombinant protein = 30
• Results and Discussion = 31
• E.coli expression vector construction = 32
• Fusion protein expression in E.coli ER2556 and one step column purification of DCD-1L from Intein fusion protein = 35
• NanoLC-ESI-MS/MS analysis for protein identification = 36
• Bioassay of recombinant LL-37 = 40
• References = 41
• CHAPTER Ⅱ Yeast episomal expression vector construction = 47
• Abstract = 48
• Introduction = 49
• Materials and Methods = 52
• Plasmid DNA and Strains = 52
• Construction of pGAPZ-E and Recombinant pGAPZ-E Harboring Reporter Genes = 52
• Subcloning of Core50 Targets into the Pichia Expression Vectors = 53
• Pichia Transformation = 54
• Pichia plasmid isolation = 54
• Protein Expression and Purification = 55
• Expression Detection of Pichia Transformants = 55
• Beta-Galactosidase Assay = 56
• Pichia Plasmid Isolation = 56
• Results and Discussion = 58
• Construction of an Episomal Expression Vectors Using PARS1 = 58
• Comparison of Episomal vs. Integral Expression of lacZ Gene = 59
• Optimization of Pichia Expression Using pGAPZ-E Vector = 60
• Episomal Expression of Structural Genomic Target Proteins = 61
• Discussion = 69
• References = 75
• CHAPTER Ⅲ Molecular cloning, expression and purification of Human AMP, LL37 and Dermcidin, in Phichia pastoris = 80
• Abstract = 81
• Introduction = 82
• Materials and Methods = 85
• Strains, media, and DNA = 85
• PCR amplification and cloning of the human LL-37 coding sequences = 86
• Construction of the pGAPZ-E/LL-37 expression vector = 86
• Peptide PAGE gel = 87
• NanoLC-ESI-MS/MS analysis for protein identification = 88
• Antimicrobial analysis of recombinant LL-37 = 89
• Results and Discussion = 90
• Construction of an expression vector = 90
• Peptide identification of LL-37 by nano-LC MS/MS analyses = 95
• Bioassay of recombinant LL-37 = 95
• References = 96
• 국문초록 = 101