Molecular biology

Intro

Kappa casein is the protein responsible for milk coagulation into cheese when it is cleaved by rennet (chymosin). If we could express this protein in yeast, we could essentially make vegan cheese.

A 2005 paper showed that the C-terminal part of the human kappa casein protein can be overexpressed in S. cerevisiae (baker’s yeast) and Pichia pastoris, and they were able to get it secreted in sufficient quantity that they could run the culture medium on a gel, and see a clear band for the protein. (“This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound.“ - valuable stuff, not just for vegan cheese!)

Our first task would be to replicate this result - ideally in the BioBricks format. Next would be to express and secrete the full-length protein. If we can get it to express and secrete in sufficient quantity, we should be able to show that you get a single band on the gel for the intact protein, but two bands after cleaving the protein with chymosin.

We can have some people working simultaneously on trying to express the other casein proteins (alpha-s1, alpha-s2, and beta). But since these are hydrophobic, we may not be able to express and secrete them without kappa-casein to hold them in suspension in a casein micelle. We can also attempt to modify the yeast lipid biosynthesis pathways to produce milk fats.

Essential reading

• Rennet coagulation of milk
• Mechanisms of Coagulation: The principles, the science and what they mean to cheesemakers
• Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris (on seafile)

Caseinomacropeptide

Kim YJ, Oh YK, Kang W, Lee EY, Park S. Production of human caseinomacropeptide in recombinant Saccharomyces cerevisiae and Pichia pastoris. J Ind Microbiol Biotechnol. 2005 Sep;32(9):402-8.

“Caseinomacropeptide is a polypeptide of 64 amino acid residues (106-169) derived from the C-terminal part of the mammalian milk k-casein. This macropeptide has various biological activities and is used as a functional food ingredient as well as a pharmaceutical compound. The gene encoding the human caseinomacropeptide (hCMP) was synthesized and expressed with an alpha-factor secretion signal in the two yeast strains, Saccharomyces cerevisiae and Pichia pastoris. The complete polypeptide of the recombinant hCMP was produced and secreted in a culture medium by both the strains, but the highest production was observed in S. cerevisiae with a galactose-inducible promoter. In a fed-batch bioreactor culture, 2.5 g/l of the recombinant hCMP was obtained from the S. cerevisiae at 97 h.” “Escherichia coli XL1-Blue (Stratagene, USA) was used for cloning and propagating genes. Host strains for recombinant hCMP were S. cerevisiae 2805 (his-, ura-) [...] For S. cerevisiae, pYIGP (containing a constitutive GAP promoter) or pYEGa (containing a galactose-inducible GAL promoter) was used.”

Yeast expression

Strain selection

The Johns Hopkins' team used YPH500

• List of commonly used lab strains
• A comparison of four strains (article access?)

Vectors

The Jons Hopkins iGEM team modified pRS400-series vectors, removing the MCS and replacing them with biobrick prefix and suffix.

Patrick says that galactose induction is the most common form of controlling heterologous expression. Common vectors are the pYES2 and pESC series vectors.

Secretion

Secretion of proteins will require a secretion signal (triggering processing through the ER, golgi and then exocytosis).

iGem Parts

• VitaYeast project, by Johns Hopkins’ 2011 iGEM team
  • Characterized promoters and terminators
• Yeast parts in the BioBricks parts registry

Other notes

pESC contains a bidirectional promoter which may allow expression of both parts of the protein without cleavage being required. How?

Casein

Patrik D says: In a bout of procrastination from grant writing, I actually stumbled upon some more really good references on casein proteins and their recombinant expression. Turns out they're not quite as insoluble as I had feared, especially if you keep the Ca concentration low enough. They've even been explored as chaperones to get other problematic proteins to fold better. There's also some papers on alpha- and beta-casein expression in E. coli and yeast, and on reconstitution of casein micelles from beta and kappa casein (beta casein is the major component in human milk).

Kappa-casein

• Kappa-casein on wikipedia
• Bovine kappa-casein precursor gene CSN3
• kappa-casein genes in other mammals

Sequence

Bovine kappa-casein is 190 AA of which the first 21 AA form a secretion signal:

MMKSFFLVVT ILALTLPFLG AQEQNQEQPI RCEKDERFFS DKIAKYIPIQ YVLSRYPSYG 
LNYYQQKPVA LINNQFLPYP YYAKPAAVRS PAQILQWQVL SNTVPAKSCQ AQPTTMARHP 
HPHLSFMAIP PKKNQDKTEI PTINTIASGE PTSTPTTEAV ESTVATLEDS PEVIESPPEI 
NTVQVTSTAV 

Post-translational modification

According to the Uniprot record, the post-translation modifications include:

• Glycosylation of six residues, all of them Threonine ((O-GalNAc)
• Phosphorylation of three residues, all of them Serine
• One disulphide bond between two cysteine residues
• One "Pyrrolidone carboxylic acid" modification to a glutamine (?)

We should investigate if any of this is important for micelle formation or micellar flocculation.

Quote from University of Illinois' Milk Composition & Synthesis Resource Library:

Phosphorylation is a ubiquitous biological process. It is involved in many regulatory mechanisms. It occurs after completion of the polypeptide chain. It can occur at Ser, Thr, Glu, Asp, His, Lys, and Tyr. Many enzymes will phosphorylate proteins with ATP, but they differ in specificity. These are called Kinases. Kinases are membrane-bound in the smooth endoplasmic reticulum and Golgi.

Caseins are phosphoproteins.

• k-Casein is not extensively phosphorylated- probably because of competition between the glycosyltransferases and the kinases for sites on the protein.
• Caseins are in multiphosphorylated forms - the same casein polypeptide chain may be phosphorylated at varying sites.
• Probably due to the tremendously high rate of protein synthesis which exceeds the phosphorylating capacity of the membrane-bound kinases.

Beta-casein expressed in E. coli has been successfully phosphorylated by co-expression of casein kinase II: Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli. The yeast kinome indicates that yeast does not include casein kinase II (CNSK2) so we would have to co-express it. I do not know if CNSK2 will also work correctly for alpha-casein (for which phosphorylation is important in micelle formation) nor for kappa-casein (where phosphorylation is probably less important), but if we cannot find more info on which caseins are phosphorylating the specific caseins, then maybe co-expressing CNSK2 is a good thing to try.

Clotting / Micellar flocculation

• Kinetics of milk coagulation: II. Kinetics of the secondary phase: micelle flocculation. (Carlson et al, 1987)
• Casein Interactions: Casting Light on the Black Boxes, the Structure in Dairy Products (Home, 1998, review)
• Studies of casein micelle structure: the past and the present (Qi, 2007, review)

If the model proposed by Home is correct, then micelle formation will require alpha, beta, kappa-casein and colloidal calcium. The model also implies that the phosphoserine residues of both alpha and kappa-casein play an important role in micelle formation.

Synthetic casein micelles

Some researchers have created synthetic casein micelles. We should study their efforts:

• Sub-structure of synthetic casein micelles (Knoop et al, 1979)
• A phosphate-induced sub-micelle-micelle equilibrium in reconstituted casein micelle systems (Slattery, 1979)
• Re-assembled casein micelles and casein nanoparticles as nano-vehicles for ω-3 polyunsaturated fatty acids (Zimet et al, 2011)

Note: None of these articles have been downloaded to seafile yet.

Genetic variants

"Substantial variation in milk coagulation properties has been observed among dairy cows. [...] the cows were genotyped for major genetic variants in the αS1- (CSN1S1), β- (CSN2), and κ-casein (CSN3) genes, revealing distinct differences in variant frequencies among breeds. [...] CSN1S1 C, CSN2 B, and CSN3 B positively affected milk coagulation, whereas CSN2 A(2), in particular, had a negative effect" [1]

Health effects of milk/cheese components

Beta casein

Populations, which consume milk containing high levels of β-casein A2 variant, have a lower incidence of cardiovascular disease and type 1 diabetes. Furthermore, consumption of milk with the A2 variant may be associated with less severe symptoms of autism and schizophrenia [2].

Cow's Milk Protein Allergy

Cow's milk allergies seem to be caused primarily by casein, and especially the alpha-s1 and beta caseins that are the major component of cow's milk. Switching to breastfeeding seems to resolve the problems, although in some instances the mother may need to avoid dairy products as well. This seems to suggest that we may be able to avoid allergic reactions by replacing the bovine caseins with the human version, or by humanizing specific epitopes on the bovine casein.

Human or bovine?

There seems to be a distinct sociological "ick" reaction against the idea of making cheese made from human casein proteins, even among those perfectly willing to consider genetically engineered cheese. On one hand, there may be significant health benefits associated with humanizing some or part of the casein proteins (probiotic effects, avoiding cow's milk allergies, etc.) On the other hand, we don't know how human caseins would behave in cheese making, and they may make the concept of an engineered cheese even less acceptable to the general public.

Suggested compromise:

• We do both the human and bovine kappa-casein at least. That should tell us whether the chymosin enzyme functions the same way on both, and whether the human kappa-casein can form micelles with bovine caseins.
• We do a little write-up on the Ethical, Legal, Societal Issues surrounding engineering with human genes and making a consumable from them (human burger, anyone?) That'll give us extra points for the iGEM competition as well.